1. Biochemistry
  2. Biophysics and Structural Biology
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Inhibition by small-molecule ligands of formation of amyloid fibrils of an immunoglobulin light chain variable domain

  1. Boris Brumshtein Is a corresponding author
  2. Shannon R Esswein Is a corresponding author
  3. Lukasz Salwinski
  4. Martin L Phillips
  5. Alan T Ly
  6. Duilio Cascio
  7. Michael R Sawaya
  8. David S Eisenberg Is a corresponding author
  1. Howard Hughes Medical Institute, UCLA, United States
  2. UCLA-DOE Institute for Genomics and Proteomics, United States
  3. UCLA, United States
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Cite as: eLife 2015;4:e10935 doi: 10.7554/eLife.10935

Abstract

Overproduction of immunoglobulin light chains leads to systemic amyloidosis, a lethal disease characterized by the formation of amyloid fibrils in patients' tissues. Excess light chains are in equilibrium between dimers and less stable monomers which can undergo irreversible aggregation to the amyloid state. The dimers therefore must disassociate into monomers prior to forming amyloid fibrils. Here we identify ligands that inhibit amyloid formation by stabilizing the Mcg light chain variable domain dimer and shifting the equilibrium away from the amyloid-prone monomer.

https://doi.org/10.7554/eLife.10935.001

eLife digest

Systemic light chain amyloidosis is a disease that occurs when individuals produce too much of an immune protein. The excess protein chains normally exist in the body as individual molecules called “monomers” or in pairs called “dimers,” and they can readily switch between these two forms. However, the monomers are also prone to forming amyloid fibrils, which are difficult to break down. Amyloid fibrils are often deposited in the heart and kidneys and can lead to organ failure and death.

Finding molecules that prevent the formation of amyloid fibrils could help to develop treatments for amyloidosis. Now, Brumshtein, Esswein et al. have screened 27 compounds to identify those that stabilize the dimer form of the protein. This would reduce the number of monomers in the body, and so reduce the number of immune proteins that can form amyloid fibrils.

The experiments identified four compounds that could stabilize the dimers, including one called methylene blue. Comparing the chemical structures of these compounds with the structures of drugs approved for medical use identified thirteen drugs. However, follow-up tests showed that only one, called sulfasalazine, reduced the formation of amyloid fibrils. Neither methylene blue nor sulfasalazine is likely to have a strong enough effect to treat amyloidosis, but they may serve as templates for future drug designs.

https://doi.org/10.7554/eLife.10935.002

Introduction

Amyloid fibrils are protein deposits often associated with disease. These deposits show common structural and biochemical characteristics, including non-covalent self-complementary fibrils, resistance to proteolysis, insolubility in aqueous solution, binding of thioflavin dye, and generation of a cross-β X-ray diffraction pattern. The spine of amyloid fibrils, termed a 'steric zipper,' is built by paired β-sheets with interdigitating side chains and serves as the scaffold for fibril extension. The inter-strand and inter-sheet distances give rise to a characteristic cross-β X-ray diffraction pattern of two perpendicular reflections at ~4.8 and ~10 Å (Geddes et al., 1968; Hobbs, 1973; Sipe and Cohen, 2000; Nelson et al., 2005). Formation of a steric zipper requires the presence of a discrete peptide segment with an amino acid sequence capable of forming a self-complimentary propagating structure. Such segments are commonly found as integral parts of globular proteins and trigger aggregation into amyloid fibrils only upon exposure to solvent through full or partial denaturation (Nelson et al., 2005).

Cases of systemic light chain amyloidosis (AL), associated with multiple myeloma, were first described in the mid-19th century; however, the etiology of the disease was not clear. In 1848, Dr. Bence-Jones discovered the disease-associated substance in urine, though it was only later identified as immunoglobulin light chains (LC) associated with amyloidosis (Kyle, 2001; 2011; Dahlin and Dockerty, 1950; Glenner et al., 1970; 1971; Glenner, 1973; Sipe et al., 2014). Systemic AL amyloidosis is characterized by overexpression of monoclonal LCs, which are able to form homo-dimers. Despite renal clearance of excess protein, the full-length LCs or their fragments still form amyloid fibril deposits in patients’ tissues, most frequently in the heart and kidneys, which result in organ failure (Falk et al., 1997; Buxbaum, 1986). Although it is clear this deposition causes organ dysfunction, much remains to be discovered about the molecular process by which immunoglobulins assemble into amyloid fibrils. It is unknown which peptide segments form steric zippers and how to block their assembly into the amyloid spine.

LCs are produced by plasma cells and their final amino acid sequence is determined by somatic recombination (Sakano et al., 1979; Marchalonis and Schluter, 1989). LCs are classified by their amino acid sequence as either κ or λ. They consist of two domains, the variable (VL) and constant (CL), which are connected by a joining segment (J). In patients, amyloid fibrils are found to include either VLs or full-length LCs (VL-J-CL), yet the ubiquitous presence of VLs indicates that this domain may be the minimal and essential unit for fibril assembly (Falk et al., 1997; Buxbaum, 1986; 1992; Olsen et al., 1998; Lavatelli et al., 2008; Vrana et al., 2009; Bodi et al., 2009).

The crystal structures of full-length LCs and structures of just their VLs both reveal homo-dimers, which resemble the light chain and heavy chain hetero-dimer of the antigen-binding fragment (Fab) of a full antibody. Fab is a hetero-dimer composed of both light and heavy chains, each with constant (CL and CH) and variable (VL and VH) immunoglobulin domains. LC homo-dimers, in which the VL domain is covalently linked to the CL domain, resemble the quaternary structure of the Fab hetero-dimer, and non-covalently linked dimers of only VLs structurally resemble the variable region of the Fab (VL and VH) (Edmundson et al., 1969; Colman et al., 1977; Firca et al., 1978). The interfaces between the variable domains of physiological hetero-dimers and pathological homo-dimers are lined with apolar residues encompassing a hydrophobic cavity capable of accommodating small molecules (Edmundson et al., 1984; 1993).

Our work focuses on a λ patient-derived VL homo-dimer named Mcg whose structure and properties were detailed in pioneering studies by Edmundson and coworkers (Edmundson et al., 1969; 1984; 1993; Firca et al., 1978). The cavity between the two VL domains in Mcg is capable of binding a wide array of ligands, including synthetic organic molecules and peptides, which form contacts with the side chains of the dimer cavity. The cavity spans most of the 24 Å-long dimer interface; it is cylindrically shaped and can be considered to constitute three sites: A, B and C (Edmundson et al., 1984). Based on the geometry of the dimer with some deviation from the original nomenclature by Edmundson et al., we designate side chains of residues Y34, Y93, D97 and F99 on one side of the cylindrical cavity as the 'A-site,' S36, Y51, E52, S91 and F101 as the 'B-site,' and Y38, Q40, V48 and Y89 as the 'C-site' (Figure 1). Some ligands bind the cavity in a sequential manner without a distinct preference for one of the sites. For example, 1-anilinonaphthalene-8-sulfonic acid binds in either the A or C-sites and can migrate between them, whereas fluorescein binds to both sites with partial occupancy. Other molecules, such as menadione, preferentially bind to only one of the sites (Edmundson et al., 1984). Regardless of binding location, this demonstrates the dimer cavity is capable of accommodating various hydrophobic and aromatic ligands.

Stereo image of the ligand-binding sites of the VL dimer.

We designate the A-site in red (residues Y34, Y93, D97 and F99), B-site in yellow (residues S36, Y51, E52, S91 and F101), and C-site in green (residues Y38, Q40, V48 and Y89).

https://doi.org/10.7554/eLife.10935.003

VLs exist in equilibrium between homo-dimers and amyloid-prone monomers. Experiments conducted in denaturing conditions indicate that reducing the stability of the monomeric state promotes amyloid fibril formation, and mutations that induce dimer disassociation or promote monomer unfolding increase the propensity to form amyloid fibrils (Bernier and Putnam, 1963; Kishida et al., 1975; Qin et al., 2007; Wetzel, 1994; Hurle et al., 1994; Brumshtein et al., 2014; Baden et al., 2008). Likewise, mutations that stabilize the structure of VLs or covalently fix VL dimers inhibit formation of amyloid fibrils. These results indicate that formation of amyloid fibrils involves two steps: VL dimer disassociation into monomers followed by partial or full unfolding. The mechanism of amyloid formation also suggests that shifting the equilibrium away from the amyloid-prone monomer by stabilizing the dimer would hinder formation of amyloid fibrils (Figure 2) (Bulawa et al., 2012; Bellotti et al., 2000).

Proposed mechanism for using ligands to hinder the aggregation of immunoglobulin VL s into amyloid fibrils.

VL s are in equilibrium between dimers and monomers in solution. Ligands may be used to stabilize the VL dimer and therefore shift the equilibrium away from amyloid-prone monomers.

https://doi.org/10.7554/eLife.10935.004

The monomer-dimer equilibrium of VLs suggests that systemic AL amyloidosis may be mitigated by binding ligands to the cavity at the VL dimer interface (Figure 2). This approach proved effective for transthyretin-related amyloidosis, another type of systemic amyloidosis for which stabilizing the quaternary state led to the development of therapeutics (Miroy et al., 1996). Upon transthyretin tetramer disassociation into amyloid-prone monomers, it forms amyloid fibrils in an acidic environment. The binding of thyroxine inhibits disassociation and subsequent amyloid formation (Baures et al., 1998). Following the same principle, a modified ligand with a disassociation constant in the nano-molar range prevents transthyretin from forming amyloid fibrils and is effective in vivo.

Here we apply structural and biochemical methods to investigate ligands that hinder amyloid formation by stabilizing the VL homo-dimer. We identify ligands that may serve as prototypes for therapies for treating LC amyloidosis and our results are consistent with a mechanism for amyloidosis that proceeds via dimer disassociation to amyloid-prone monomers (Qin et al., 2007; Brumshtein et al., 2014).

Results

Identification of ligands that inhibit formation of amyloid fibrils. Based on the previous work of Edmundson et al., we screened a panel of 27 hydrophobic and aromatic ligands for an inhibitory effect on the formation of amyloid fibrils (Figure 3) (Edmundson et al., 1976; 1984; 1993). Such ligands may stabilize VL dimers and decrease disassociation into amyloid-prone monomers. Varying concentrations of ligands up to 1 mM were used to evaluate the effect of complexes on formation of amyloid fibrils. Formation of amyloid fibrils was monitored using both ThT fluorescence assays and EM micrographs. Molecules that showed an inhibitory effect with concentrations under 1 mM were 8-anilino-1-naphthalene sulfonic acid [5], methylene blue [10], Chicago Sky Blue 6B [14] and Phenol Red [20] (Figure 3B).

Panel of ligands analyzed with ThT assays and EM for their ability to inhibit VL amyloid formation.

(A) Preliminary molecules screened. 1. 1,2-naphthoquinone. 2. 1,4-naphthoquinone. 3. 5-hydroxy-1,4-naphthoquinone. 4. quinone. 5. 8-anilinonaphthalene-1-sulfonic acid. 6. methoxy-2-naphthyl-propionic acid. 7. dopamine. 8. L-phenylalanine. 9. L-tryptophan. 10. methylene blue. 11. eosin Y. 12. rhodamine 6G. 13. Basic Blue 12. 14. Chicago Sky Blue 6B. 15. 8-aminonaphthalene-1,3,6-trisulfonic acid. 16. Basic Blue 41. 17. dimenhydrinate. 18. stictic acid. 19. 6-carboxyfluorescein. 20. phenol red. 21. R-(-)-apomorphine. 22. sulforhodamine 101. 23. fluorescein methyleneiminodiacetic acid. 24. rhodamine B. 25. tetracycline. 26. doxycycline. 27. (−)-epigallocatechin gallate. (B) Molecules showing an inhibitory effect on the formation of amyloid fibrils during the preliminary screen. (C) Molecules identified in a DrugBank search using the most effective candidates from the preliminary screen and a similarity threshold of 0.3. Search using phenol red: 28. diethylstilbestrol (0.4), 29. sulindac (0.4), 30. tamoxifen (0.4), 31. acitretin (0.4). Search using Chicago Sky Blue 6B: 32. suramin (0.4), 33. Bumetanide (0.3), 34. sulfasalazine (0.3), 35. sulfadoxine (0.3), 36. amsacrine (0.3), 37. methotrimeprazine (0.3). Search using methylene blue: 38. clofazimine (0.3), 39. primaquine (0.3), 40. imiquimod (0.3).

https://doi.org/10.7554/eLife.10935.005

Methylene blue, Chicago Sky Blue 6B, and Phenol Red (10, 14 and 20) were used to search DrugBank for approved biomedical compounds resembling their chemical structures and properties with a similarity threshold value of 0.3 (Law et al., 2014). The search identified 13 compounds that were tested for an amyloid inhibitory effect by means of ThT assays and EM analysis (Figure 3C). Of these 13 molecules from DrugBank, only sulfasalazine [34] showed an inhibitory effect on formation of amyloid fibrils. Among all the molecules tested, including the ligands initially screened and the subsequent approved biomedical compounds, the two that show the most potent effect are methylene blue [10] and sulfasalazine [34]; both hinder amyloid fibril formation at effective concentrations of 0.1 and 0.5 mM respectively (Figure 4).

Thioflavin T fibril formation assays and electron micrographs of Mcg VLs in the presence of various concentrations of methylene blue and sulfasalazine.

Averaged fluorescence readings are based on three repeated ThT assays. Error bars are not shown for clarity. The EM micrographs have a scale bar of 0.5 μm. (A) Mcg alone. (B) Mcg with various concentrations of methylene blue. (C) Mcg with various concentrations of sulfasalazine. As the concentration of ligand is increased, the ability of VLs to form amyloid fibrils is hindered, as indicated by a decrease in ThT assay fluorescence and inability to detect fibrils with EM. The ligand concentration that effectively inhibited amyloid formation in these assays was 500 μM for methylene blue and 250 μM for sulfasalazine.

https://doi.org/10.7554/eLife.10935.006

Equilibrium dialysis binding of methylene blue and sulfasalazine. Equilibrium dialysis was used to assess the binding constants of methylene blue and sulfasalazine to Mcg. Measured concentrations were fit to the corresponding model equations and their curves were represented as binding and Scatchard plots (Figure 5) (Scatchard, 1949; Spitzer and McDonald, 1956). The constants were derived from a least squares fit of equations to data and are given in Table 1. Although both methylene blue and sulfasalazine bind to Mcg, the Scatchard plots indicate that binding proceeds through somewhat different pathways: methylene blue shows positive cooperative binding, signifying at least two sites with different binding constants, while sulfasalazine shows no cooperativity and suggests an additional, non-specific binding site (Figure 5). The best fit for the sulfasalazine-binding data was achieved using a model for two identical, independent binding sites per VL dimer, followed by non-specific binding.

Binding of ligands to Mcg VLs.

Binding curves (top) and Scatchard plots (bottom) of ligand binding determined from equilibrium dialysis experiments. Each curve represents binding equations fit to the data by least squares. Binding constants were derived from the fit equations (see Table 1). Vertical bars represent the standard errors of the mean from independently repeated experiments. [B], [L], and [U] are bound, total, and unbound concentrations of ligand in μM. (A) Methylene blue binding to Mcg. Rhombs show means for 3 independent experiments performed with 1.0 mg/ml Mcg. Circles show means for 5 independent experiments performed with 0.5 mg/ml Mcg. Notice the sigmoidal shape of the binding plot and concave shape of the Scatchard plot indicating cooperative binding. (B) Sulfasalazine binding to Mcg. Rhombs show means for 3 independent experiments performed with 5.0 mg/ml Mcg. Circles show means for 3 independent experiments performed with 2.5 mg/ml Mcg. Notice the saturated binding and a down-curving shape of the Scatchard plot indicating one binding constant followed by non-specific binding.

https://doi.org/10.7554/eLife.10935.007
Table 1

Disassociation constants of ligands that bind to the Mcg VL dimer.

https://doi.org/10.7554/eLife.10935.008
Ligand<K1>
[μM] (SEM)
<K2> or KNS
[μM] (SEM)
NBinding modelEquations fit
([Y]: bound ligand,
[L]: free ligand,
[R]: receptor)
methylene blue207 (35)21 (4)8Two positively
cooperative sites
R+L⇔ RL+L ⇔ RL2
(1) [Y]=([L]*[R]/K1)*(1+ [L]/K2))
(2) [R]=[R0]/(1+[L]/K1+[L]2/(K1*K2))
sulfasalazine698 (105)21 (8) (non-specific
binding)
6Two equivalent
+ non-specific sites
R+L ⇔ RL
(3) [Y]=[R0]*[L]/(K1+[L]) + KNS*[L]
SEM – standard error of the mean; N – number of independent experiments
KNS – nonspecific binding

Crystal structures of methylene blue and sulfasalazine bound to the VL dimer. In the crystal structures of Mcg with methylene blue and sulfasalazine, the ligands bind at the cavity between the two VL domains (Figure 6, Table 2). In the structure of Mcg with methylene blue, one ligand is bound to the A-site of the dimer. This differs from equilibrium dialysis results in solution, which indicate at least two methylene blue binding sites. The structure of Mcg with sulfasalazine indicates two symmetry-related molecules bound in the hydrophobic cavity of the dimer with both ligands simultaneously binding to three sites of the VL dimer cavity.

Crystal structures of Mcg binding methylene blue (PDB 5ACM) and Mcg binding sulfasalazine (PDB 5ACL).

(A) Mcg and methylene blue. Methylene blue binds the cavity between the VL domains. The sulfate ion binds in the hydrophobic cavity at S36 and may block the secondary methylene blue binding site. (B) Mcg and sulfasalazine. Two symmetry-related sulfasalazine molecules bind in the hydrophobic cavity between the VL domains, simultaneously occupying all three sites: A, B, and C. (C) Overlay of the structures from A and B for comparison and annotation of A-B-C binding sites.

https://doi.org/10.7554/eLife.10935.009
Table 2

Statistics of X-ray data collection and atomic refinement. Values in parentheses are for the outermost shell of data. Each structure was derived from a single crystal.

https://doi.org/10.7554/eLife.10935.010
Mcg-methylene blue
PDB 5ACM
Mcg-sulfasalazine
PDB 5ACL
Space groupP21P21212
Cell dimensions
a, b, c (Å)39.2, 31.1, 73.631.8, 74.6, 39.3
α, β, γ (°)90, 90.1, 9090, 90, 90
Resolution (Å)39.2-1.0 (1.07)29.3-1.5 (1.53)
Rsym (%)7.2 (59.1)5.5 (52.9)
I/σ(I) 9.8 (2.4)18.7 (3.7)
Completeness (%)88.6 (81.8)97.9 (83.2)
Redundancy3.5 (3.5)6.9 (5.9)
No. reflections259214107913
Rwork/Rfree (%)11.1/12.215.9/18.7
No. atoms
Protein1610845
Ligand/ion3633
Water210134
Average B-factors (Å2)16.820.4
R.m.s deviations
Bond lengths (Å)0.050.02
Bond angles (º)3.272.04
Crystallization conditions0.2 M NH4Cl, 2.2 M (NH4)2SO4, 0.5 M methylene blue0.2 M Tri-potassium citrate, 2.2 M
(NH4)2SO4, 0.5 M sulfasalazine

Effect of ligand binding on quaternary state. We used analytical ultracentrifugation to assess whether ligand binding shifts the equilibrium of Mcg towards more dimers or whether ligands bind to monomers as well. The wavelengths used for optical density readings of methylene blue (550 nm) and sulfasalazine (420 nm) did not overlap with protein absorbance. Distributions of molecular weights were fit to the absorbance data, thus permitting detection of only the apparent molecular weights of the protein in complex with ligand (Figure 7, Table 3). The observed molecular weights indicate that ligands bind only to VL dimers. For methylene blue, the measured molecular weight was slightly larger than expected; therefore, we performed an additional ultracentrifugation meniscus depletion experiment to detect possible monomers. The experiment identified no methylene blue bound to VL monomers.

Analytical ultracentrifugation data for Mcg with methylene blue and Mcg with sulfasalazine.

(A) Absorbance and residuals for 80 μM Mcg with 100 μM methylene blue at 22,000 rpm. (B) Absorbance and residuals for 80 μM Mcg with 150 μM methylene blue at 22,000 rpm. (C) Dimer meniscus depletion - absorbance and residuals for 80 μM Mcg with 100 μM methylene blue at 35,000 rpm. (D) Dimer meniscus depletion - absorbance and residuals for 80 μM Mcg with 150 μM methylene blue at 35,000 rpm. (E) Absorbance and residuals for 200 μM Mcg with 300 μM sulfasalazine at 22,000 rpm. (F) Absorbance and residuals for 200 μM Mcg with 500 μM sulfasalazine at 22,000 rpm. Measurements were taken after samples reached equilibrium and the distribution of protein along the cells did not change. Residuals show the corresponding error of the fit of the model to the data.

https://doi.org/10.7554/eLife.10935.011
Table 3

Apparent VL molecular weights and ligand binding derived from analytical ultracentrifugation.

https://doi.org/10.7554/eLife.10935.012
Speed (rpm)Concentration of Mcg (μM)LigandConcentration of ligand (μM)Apparent molecular weight (Da)Oligomer state
22,00080methylene blue10027,342 ± 268*Dimer + multimer
15026,507 ± 225*Dimer + multimer
35,00080methylene blue10023,609 ± 212*Dimer
15023,397 ± 152*Dimer
22,000200sulfasalazine30021,989 ± 571*Dimer
50021,821 ± 345*Dimer
  1. *Standard error.

Discussion

The identification of ligands that inhibit LC amyloid formation by stabilizing the VL dimer has therapeutic implications for systemic light chain amyloidosis. By utilizing the known ability of ligands to bind the hydrophobic cavity of VL dimers, we prevent dimer disassociation into monomers and thus hinder amyloid formation (Edmundson et al., 1984; 1993). A similar approach was used to design therapies for transthyretin-related amyloidosis by stabilizing the tetrameric state of the protein (Miroy et al., 1996). Initial screening for ligands that hinder VL amyloid formation revealed four ligands showing this effect, with methylene blue as the strongest binding ligand. A subsequent search in the DrugBank aimed to identify similarly effective ligands that are already approved for medical use.

Since the emission spectra of ThT partly overlaps with the absorption spectra of the ligands, both ThT assays and EM analysis were necessary to confirm the ability of the ligands to inhibit amyloid formation. Analysis of electron micrographs and ThT assays indicates that increasing the concentration of methylene blue or sulfasalazine hinders the formation of amyloid fibrils. One of the molecules from our screen, epigallocatechin gallate, was recently suggested to hinder the formation of amyloid fibrils, yet in our experiments it did not show any effect (Pelaez-Aguilar et al., 2015).

Though both of the ligands bind Mcg and induce amyloid hindering effects, the mechanism of binding slightly differs. Methylene blue binds through positive cooperativity. This result indicates the presence of two binding sites, one strong and one weak, where binding to the second site begins only after the first site is fully occupied. Scatchard plots for sulfasalazine show that the two specific binding sites on Mcg are identical with the same disassociation constant and that this specific ligand binding is followed by non-specific binding.

We addressed the difference in the binding mechanisms of methylene blue and sulfasalazine to Mcg in solution by crystal structures and analytical ultracentrifugation. The crystal structure of Mcg with methylene blue shows binding at the A-site of the VL dimer cavity and coordination by the side chains of residues Y34, Y93, D97 and F99. The crystal belongs to space group P21 with one dimer per asymmetric unit, and the symmetry between the two VL domains of Mcg is broken by the presence of a sulfate ion bound to only one of the VL domains (Figure 6A). Analytical ultracentrifugation analysis of the soluble quaternary state indicates the dimer as the most prevalent form of the complex versus monomer without a bound ligand (Brumshtein et al., 2014). We also observed a small fraction of oligomers with molecular weights larger than dimers, as confirmed by the meniscus depletion experiment. The meniscus depletion distributes quaternary states along the path length of the sample cell during analytical centrifugation, thus verifying which state accounts for the deviation in apparent molecular weight. The most significant outcome is the complete absence of soluble monomer in complex with the ligand, which verifies our conjecture that the ligands bind and stabilize dimers. In the case of sulfasalazine, to confirm binding specifically to soluble dimers, the analytical ultracentrifugation experiments identified dimers as the only quaternary state of the ligand-bound complex.

The reconciliation of our three experiments on the binding of methylene blue to Mcg VLs suggests blocking of the secondary binding site in the crystals. The three experimental findings are: cooperative binding of methylene blue as measured by equilibrium dialysis, binding of the ligand to the A-site in the crystal structure, and the absence of monomeric VL in complex with ligand. Taken together, these observations indicate that the secondary binding site of methylene blue may not be available in the crystalline state, but available in solution. Secondary binding to either the B or C-sites may require conformational changes that are less favorable in crystals or induce the higher-order assembly of complexes larger than dimers.

The crystal structure of Mcg with sulfasalazine explains why only one disassociation constant for specific binding was measured. Crystals of the complex belong to space group P21212 with two symmetry-related ligands bound per Mcg dimer, and thus one ligand bound per VL in the asymmetric unit (Figure 6B). Therefore, the equivalence of the two sulfasalazine ligands in the crystal explains why only one disassociation constant was measured by means of equilibrium dialysis. The crystal structure shows that each of the sulfasalazine ligands occupy all three A-B-C-sites simultaneously; each ligand binds to the A and B-sites on one of the VLs composing the dimer, while C-site binding is on the symmetry-related VL. This contrasts with methylene blue, which is bound only to the A-site. Despite differences in the exact orientation of the side chains where the ligands bind, both ligands bind in the cavity of the Mcg dimer (Figure 6C).

Although our experiments were performed using only VLs rather than full-length LCs, the ligand-binding site between VLs is maintained in full-length LCs. While full-length LCs are disulfide linked at their CL C-termini, crystal structures indicate that the conformation of VLs is independent of connected constant domains (Ely et al., 1989; Terzyan et al., 2003; Makino et al., 2007). In full-length LCs, the joining (J) segment between VLs and CLs provides the flexibility for VLs to maintain the same hydrophobic cavity as that seen in the VL homo-dimer alone. This indicates the strategy of using ligands to stabilize the VL dimer and hinder amyloid fibril formation may also be effective for full-length homo-dimers.

The ability of ligands to hinder the aggregation of VLs into amyloid fibrils by stabilizing VL dimers indicates a promising approach for treatment of systemic light chain amyloidosis disease. While the light chain amino acid sequence differs between patients, the characteristic hydrophobic cavity of the homo-dimer formed by overexpressed LCs is conserved (Bodi et al., 2009; Bork et al., 1994; Edmundson et al., 1976; Solomon and Weiss, 1995). Although our experiments were performed using a single VL variant, LCs of both λ and κ maintain a similar tertiary structure and hydrophobic cavity (Qin et al., 2007; Wetzel, 1994; Brumshtein et al., 2014; Bellotti et al., 2000; Fink, 1998; Khurana et al., 2001). This conserved hydrophobic pocket indicates a possible target for designing medications that inhibit LC amyloidosis. Although methylene blue and sulfasalazine may not bind tightly enough to be directly repurposed as treatments for the disease, these ligands may serve as prototypes for development of effective medications. As indicated by the ability of these molecules to inhibit amyloid fibril formation, this approach may result in alleviated symptoms for systemic light chain amyloidosis patients.

Materials and methods

Preparation of recombinant proteins

Protein samples were prepared as previously described (Brumshtein et al., 2014). In summary, the VL of the pathologic LC homo-dimer Mcg was expressed in E. Coli, purified in denaturing conditions, refolded, and concentrated.

Identification of ligands that impede amyloid fibril formation

Following previous research demonstrating the ability of ligands to bind in the hydrophobic cavity between the two VL domains of the dimer, aromatic and other hydrophobic ligands were screened for an inhibitory effect on the formation of amyloid fibrils (Edmundson et al., 1984;1993). Amyloid formation was monitored with Thioflavin T assays and electron microscopy analysis, and we used molecules that inhibited amyloid fibril formation at concentrations below 1 mM to search DrugBank to identify biomedical compounds with a similar structure that would potentially inhibit the formation of amyloid fibrils (Law et al., 2014). We selected structures in DrugBank with a similarity threshold of 0.3. Search filters were not applied to any other parameters. We dissolved the identified molecules in either water or 20% DMSO for subsequent use in assays of amyloid formation.

Amyloid fibril formation assays

Thioflavin (ThT) assays of Mcg amyloid formation were performed in acidic conditions as previously described (Bellotti et al., 2000). Assays were conducted in the presence of varying concentrations of the ligands (1.0, 0.50, 0.25, 0.12, 0.05, and 0.03 mM) at 37°C and constant 300 rpm shaking with Teflon balls with 0.125 inch radii as stirrers. ThT fluorescence spectra were recorded at 440/480 nm excitation/emission wavelengths (Wall et al., 1999).

Electron microscopy (EM)

Samples from the ThT fibril formation assays were diluted with water to 10% v/v and applied onto copper grids with formvar-carbon coating (Ted Pella, Inc., Redding, CA, Cat. No. 01810). Negative staining was performed with 2% w/v uranyl acetate and images were collected by means of a Tecnai T12 electron microscope at 120 kV with a Gatan CCD camera.

Equilibrium dialysis binding

Binding constants of the ligands with Mcg were derived using Rapid Equilibrium Dialysis plates with a membrane cutoff of 8 kDa (Thermo Scientific Inc., Cat. No. 90006) (Waters et al., 2008). Methylene blue at concentrations ranging from ~1–200 μM with 0.5 and 1.0 mg/ml of Mcg, and sulfasalazine at concentrations ranging from ~100–5000 μM with 2.5 and 5.0 mg/ml of Mcg were incubated for 72 hours with constant agitation in standard laboratory conditions. Following incubation, samples were collected from the membrane and reference cells and final concentrations were measured by spectrophotometry.

Crystal structure determination

Co-crystallization trials were set up with a Mosquito micro-crystallization robot, using a solution of 20 mg/ml Mcg with 1 mM methylene blue or sulfasalazine ligands. All crystals appeared in the presence of sulfate or phosphate anions. Crystals were cryo-protected with 25% glycerol and X-ray data were collected at the 24-ID-C beamline of the Advanced Photon Source (Mcg-methylene blue) or with a Rigaku Raxis4++ with a HCT detector (Mcg-sulfasalazine). X-ray diffraction data were processed with XDS package (Kabsch, 2010). Initial phases were obtained by means of molecular replacement using PDB 3MCG as the initial model and structures were refined with Refmac5 (Murshudov et al., 1997). Graphics were rendered with PyMol and refined atomic models were deposited into the Protein Data Bank (PDB) (DeLano, 2002).

Analytical ultracentrifugation

Sedimentation equilibrium experiments were performed on Mcg in the presence of methylene blue and sulfasalazine. Equilibrium runs were performed at 20°C in a Beckman Optima XL-A analytical ultracentrifuge using absorption optics at 500 nm for methylene blue and 420 nm for sulfasalazine. Samples were at concentrations of 1 mg/ml of Mcg with 100 and 150 μM of methylene blue, and 1 and 2.5 mg/ml of Mcg with 300 and 500 μM sulfasalazine. The concentrations of the protein and ligands were chosen to obtain optical density readings in the linear range of the spectrophotometer yet with measurable quaternary state densities of the complexes.

Sedimentation equilibrium profiles were measured at speeds of 10,000, 14,000 and 22,000 rpm in a An-60 Ti rotor. To validate the deviation of the apparent molecular weight of Mcg with methylene blue, a set of experiments analyzing the meniscus depletion of the Mcg dimer were performed at 35,000 rpm. The data were fit with a non-linear least squares double exponential fit using the Beckman Origin-based software (Version 3.01) (Cohn and Edsall, 1943; Laue et al., 1992).

References

  1. 1
  2. 2
    Discovering transthyretin amyloid fibril inhibitors by limited screening
    1. PW Baures
    2. SA Peterson
    3. JW Kelly
    (1998)
    Bioorganic & Medicinal Chemistry 6:1389–1401.
  3. 3
  4. 4
  5. 5
  6. 6
  7. 7
  8. 8
  9. 9
  10. 10
    Mechanisms of disease: monoclonal immunoglobulin deposition. amyloidosis, light chain deposition disease, and light and heavy chain deposition disease
    1. J Buxbaum
    (1992)
    Hematology/oncology Clinics of North America 6:323–346.
  11. 11
    Proteins, Amino Acids and Peptides as Ions and Dipolar Ions
    1. E Cohn
    2. J Edsall
    (1943)
    Density and apparent specific volume of proteins, Proteins, Amino Acids and Peptides as Ions and Dipolar Ions, New York, Reinhold Publishing Corporation.
  12. 12
  13. 13
    Amyloid and myeloma
    1. DC Dahlin
    2. MB Dockerty
    (1950)
    The American Journal of Pathology 26:581–593.
  14. 14
    The PyMol Molecular Graphics System, version  Version 1.7.4
    1. W DeLano
    2. Schrödinger, Llc
    (2002)
    The PyMol Molecular Graphics System.
  15. 15
    A crystallographic investigation of the mcg myeloma protein. ANL-7635
    1. AB Edmundson
    2. MK Wood
    3. M Schiffer
    4. KD Hardman
    5. CF Ainsworth
    (1969)
    ANL pp. 283–285.
  16. 16
    Conformational isomerism, rotational allomerism, and divergent evolution in immunoglobulin light chains
    1. AB Edmundson
    2. KR Ely
    3. EE Abola
    4. M Schiffer
    5. N Panagiotopoulos
    6. HF Deutsch
    (1976)
    Federation Proceedings 35:2119–2123.
  17. 17
    A search for site-filling ligands in the mcg bence-jones dimer: crystal binding studies of fluorescent compounds
    1. AB Edmundson
    2. KR Ely
    3. JN Herron
    (1984)
    Molecular Immunology 21:561–576.
  18. 18
  19. 19
  20. 20
  21. 21
  22. 22
  23. 23
  24. 24
  25. 25
  26. 26
  27. 27
    An ABC of amyloid
    1. JR Hobbs
    (1973)
    Proceedings of the Royal Society of Medicine 66:705–710.
  28. 28
    A role for destabilizing amino acid replacements in light-chain amyloidosis
    1. M Hurle
    2. L Helms
    3. L Li
    4. W Chan
    5. R Wetzel
    (1994)
    Proceedings of the National Academy of Sciences of the United States of America 91:5446–5450.
    https://doi.org/10.1073/pnas.91.12.5446
  29. 29
    Xds
    1. W Kabsch
    (2010)
    Acta Crystallographica Section D Biological Crystallography 66:125–132.
    https://doi.org/10.1107/S0907444909047337
  30. 30
  31. 31
    A type kappa bence jones protein containing a cysteinyl residue in the variable region
    1. F Kishida
    2. T Azuma
    3. K Hamaguchi
    (1975)
    Journal of Biochemistry 77:481–491.
  32. 32
    Amyloidosis: a convoluted story
    1. RA Kyle
    (2001)
    British Journal of Haematology 114:529–538.
  33. 33
  34. 34
    Analytical Ultracentrifugation in Biochemistry and Polymer Science
    1. T Laue
    2. B Shah
    3. T Ridgeway
    4. S Pelletier
    (1992)
    Computer-Aided Interpretation of Analytical Sedimentation Data for Proteins, Analytical Ultracentrifugation in Biochemistry and Polymer Science, Cambridge, Great Britain, The Royal Society of Chemistry.
  35. 35
  36. 36
  37. 37
  38. 38
    Evolution of variable and constant domains and joining segments of rearranging immunoglobulins
    1. JJ Marchalonis
    2. SF Schluter
    (1989)
    Faseb Journal 3:2469–2479.
  39. 39
  40. 40
  41. 41
  42. 42
  43. 43
  44. 44
  45. 45
  46. 46
  47. 47
  48. 48
  49. 49
    Structural and functional properties of human lambda-light-chain variable-region subgroups
    1. A Solomon
    2. DT Weiss
    (1995)
    Clinical and Diagnostic Laboratory Immunology 2:387–394.
  50. 50
  51. 51
  52. 52
  53. 53
  54. 54
  55. 55

Decision letter

  1. Jeffery W Kelly
    Reviewing Editor; Scripps Research Institute, United States

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for submitting your work entitled "Inhibition by small-molecule ligands of formation of amyloid fibrils of variable domains of IgG light chains" for peer review at eLife. Your submission has been favorably evaluated by Randy Schekman (Senior editor) and two reviewers, one of whom (Jeffery W. Kelly) is a member of our Board of Reviewing Editors.

The Reviewing editor has drafted this decision letter to help you prepare a revised submission.

Both the reviewer and the Reviewing editor agree that the authors have demonstrated an important proof-of-principle regarding the potential utility of a dimer stabilizing therapeutic approach for light chain amyloidosis, which has long being theorized for systemic AL amyloidosis (see Bellotti et al., J Struct Biol 2000).

We seek a minor revision from the authors: addressing all the questions/suggestions posed in the two reviews and two minor comments below.

Reviewer #1:

The manuscript by Brumshtein et al. entitled "Inhibition by small-molecule ligands of formation of amyloid fibrils of variable domains of Ig light chains" is an excellent paper that merits serious consideration for publication in eLife. The authors have accomplished a feat that many groups have tried to accomplish previously – i.e. to stabilize the light chain variable domain dimer which is non-amyloidogenic, inhibiting dimer dissociation to an amyloidogenic monomer.

While the light chain dimer sequence varies between patients, the hydrophobic cavity at the variable domain dimer-dimer interface is conserved in sequence. The light chain variable domain dimer dissociates, misfolds and aggregates driving degenerative disease phenotypes. Thus, the authors screened a library of small molecules previously implicated to bind at the variable domain dimer interface. Analysis of electron micrographs and thioflavin T assays reveals that increasing the concentration of methylene blue or sulfasalazine hinders the formation of light chain aggregation. Two ligands bind to each dimer, methylene blue binding with positive cooperativity, whereas sulfasalazine binds non-cooperatively. Analytical ultracentrifugation is consistent with ligand binding to and stabilization of the dimer. In the crystal structure only one molecule of methylene blue binds / dimer vs. two molecules of sulfasalazine / dimer. While these two ligands do not bind tightly enough to become drug candidates, they convincingly demonstrate proof-of-principle that molecules that bind to the non-amyloidogenic variable domain light chain dimer prevent light chain aggregation driving light chain amyloidosis. This data provides the incentive for a commercial organization to make a non-amyloidogenic light chain dimer stabilizer.

Can the authors please comment on whether this strategy could be useful for inhibition of the amyloidogenesis from the disulfide-linked full-length light chain dimer, wherein each chain comprises 1 constant and 1 variable domain, i.e., is the same binding site conserved?

Reviewer #1 (Minor Comments):

1) The first sentence of the Introduction is a run-on sentence that should be rewritten as two sentences: "Amyloid fibrils are protein deposits, which are often associated with disease and show common structural and biochemical characteristics, including the aggregation into proteins into non-covalent self-complementary fibrils, resistance to proteolysis, insolubility in aqueous solution, binding of thioflavin dye, and generation of a cross-beta X-ray diffraction pattern."

2) That dimer dissociation and partial monomer denaturation drives variable domain aggregation was shown by Wetzel et al. (early 80's) and this literature should be referenced.

3) In Figure 2 it would be best to depict the dissociated amyloidogenic monomers as having a shape other than that in the non-amyloidogenic dimer.

Reviewer #2:

Brumshtein and colleagues report the identification of ligands (including approved biomedical compounds) that bind and stabilize dimers of a human amyloidogenic immunoglobulin λ light chain (Mcg), thereby inhibiting amyloid formation in vitro.

This is an important proof-of-principle study about the potential utility of such a therapeutic approach, which is currently being pursued for ATTR amyloidosis and which has long being theorized also for systemic AL amyloidosis (see Bellotti et al., J Struct Biol 2000).

As discussed by the authors, each patient with systemic AL amyloidosis has a unique immunoglobulin light chain sequence. However, the study is focused on an individual amyloidogenic immunoglobulin λ light chain (Mcg). Whereas this is acceptable for a proof-of-principle study (even though analysis of other amyloidogenic light chains, including also κ light chains, would represent a significant improvement) this fact should be better discussed in the manuscript, and should also be reflected in the title and in the Abstract.

Reviewer #2 (Minor Comments):

1) Title: "Inhibition by small-molecule ligands of formation of amyloid fibrils of variable domains of IgG light chains". The abbreviation IgG (immunoglobulin of G type) is inappropriate in this case and should be replaced with immunoglobulin. To take into account the above mentioned issue of having investigated only Mcg, a new suggested title could be: "Inhibition by small-molecule ligands of formation of amyloid fibrils of variable domains of Mcg immunoglobulin light chains". This would also be in line with the work done by the group of Edmundson on Mcg, where the focus on Mcg is reflected in the titles of the different papers.

2) Introduction: "[…] including the aggregation into proteins into non covalent self complementary fibrils […]". This is not clear, and should be rephrased.

3) Introduction: "Systemic light chain amyloidosis, frequently associated with multiple myeloma, was first described in 1848 when Dr. Bence Jones discovered a disease associated substance in urine […]". Systemic AL amyloidosis is only occasionally associated with multiple myeloma and was not discovered first by Dr. Bence Jones (see Kyle, Amyloid 2011).

4) Introduction: "This disease is characterized by overexpression of monoclonal LCs, which are able to form homo dimers termed Bence Jones proteins […]". The use of Bence Jones protein referred to homo dimers of monoclonal light chain is inappropriate.

5) Introduction: "LCs are produced by white blood cells […]" is too generic. Use Plasma cells instead of white blood cells.

6) Introduction: "The mechanism of amyloid formation also suggests that shifting the equilibrium away from the amyloid prone monomer by stabilizing the dimer would hinder formation of amyloid fibrils (Figure 2). (Bulawa et al., 2012)." Quote also Bellotti et al., J Struct Biol 2000.

7) Introduction: "The monomer dimer equilibrium of VLs suggests that systemic LC amyloidosis […]". The use of systemic AL amyloidosis, according to international guidelines on nomenclature, would be preferable (see Sipe et al., Amyloid 2014).

8) Materials and methods: The addition of a short paragraph on the in silico search performed in the Drugbank website, including which type of search (ChemQuery? Other?), search options and applied filters, would be useful.

9) Figure 4: Replace 0.5 μM with 0.5 μm in the scale bar description. Define FI in the legend. Add units of measure in all y axes of panels A, B and C.

10) Figure 5: Define [B], [L] and [B]/[U] in the legend. Add units of measure in all x and y axes of panels A and B.

11) Figure 7: Add units of measure in all y axes of panels A to F.

12) Discussion: "The most significant outcome is the complete absence of soluble monomer in complex with the ligand, which verifies our conjecture that the binding of ligands to VLs induces dimerization". Does binding of ligands to VLs induce dimerization or do ligands bind and stabilize dimers?

13) Discussion: "While the light chain amino acid sequence differs between patients, the characteristic hydrophobic cavity of the VL homo dimer formed by overexpressed LCs is conserved". This point is central to the more broaden applicability of the therapeutic approach suggested in this study. This point should be better explained and appropriate references should be quoted to circumstantiate this claim.

https://doi.org/10.7554/eLife.10935.013

Author response

Reviewer #1:

[…] Can the authors please comment on whether this strategy could be useful for inhibition of the amyloidogenesis from the disulfide-linked full-length light chain dimer, wherein each chain comprises 1 constant and 1 variable domain, i.e., is the same binding site conserved?

We added a paragraph explaining the topic before the last paragraph of the Discussion. The modified text is: “Although our experiments were performed using only VLs rather than full-length LCs, the ligand-binding site between VLs is maintained in full-length LCs. […] This indicates the strategy of using ligands to stabilize the VL dimer and hinder amyloid fibril formation may also be effective for full-length homo-dimers.”

Reviewer #1 (Minor Comments):

1) The first sentence of the Introduction is a run-on sentence that should be rewritten as two sentences: "Amyloid fibrils are protein deposits, which are often associated with disease and show common structural and biochemical characteristics, including the aggregation into proteins into non-covalent self-complementary fibrils, resistance to proteolysis, insolubility in aqueous solution, binding of thioflavin dye, and generation of a cross-beta X-ray diffraction pattern."

We split the run-on sentence into two and polished them. The modified text is: “Amyloid fibrils are protein deposits often associated with disease. These deposits show common structural and biochemical characteristics, including non-covalent self-complementary fibrils, resistance to proteolysis, insolubility in aqueous solution, binding of thioflavin dye, and generation of a cross-β X-ray diffraction pattern.”

2) That dimer dissociation and partial monomer denaturation drives variable domain aggregation was shown by Wetzel et al. (early 80's) and this literature should be referenced.

We referenced Wetzel et al.

3) In Figure 2 it would be best to depict the dissociated amyloidogenic monomers as having a shape other than that in the non-amyloidogenic dimer.

We prefer to keep the original figure, since monomers are in equilibrium with dimers in solution and the tertiary structure is maintained unless the monomer proceeds to unfold and form amyloid fibers.

Reviewer #2:

[…] As discussed by the authors, each patient with systemic AL amyloidosis has a unique immunoglobulin light chain sequence. However, the study is focused on an individual amyloidogenic immunoglobulin λ light chain (Mcg). Whereas this is acceptable for a proof-of-principle study (even though analysis of other amyloidogenic light chains, including also κ light chains, would represent a significant improvement) this fact should be better discussed in the manuscript, and should also be reflected in the title and in the Abstract.

We modified the title to: “Inhibition by small-molecule ligands of formation of amyloid fibrils of an immunoglobulin light chain variable domains”

We modified the last sentence of the Abstract to: “Here we identify ligands that inhibit amyloid formation by stabilizing the Mcg light chain variable domain dimer and shifting the equilibrium away from the amyloid-prone monomer.”

We added an explanation to the last paragraph of the Discussion. The new text is: “The ability of ligands to hinder the aggregation of VLs into amyloid fibrils by stabilizing VL dimers indicates a promising approach for treatment of systemic light chain amyloidosis disease. […] As indicated by the ability of these molecules to inhibit amyloid fibril formation, this approach may result in alleviated symptoms for systemic light chain amyloidosis patients.”

Reviewer #2 (Minor Comments):

1) Title: "Inhibition by small-molecule ligands of formation of amyloid fibrils of variable domains of IgG light chains". The abbreviation IgG (immunoglobulin of G type) is inappropriate in this case and should be replaced with immunoglobulin. To take into account the above mentioned issue of having investigated only Mcg, a new suggested title could be: "Inhibition by small-molecule ligands of formation of amyloid fibrils of variable domains of Mcg immunoglobulin light chains". This would also be in line with all the work done by the group of Edmundson on Mcg, where the focus on Mcg is reflected in the titles of the different papers.

See above.

2) Introduction: "[...] including the aggregation into proteins into non covalent self complementary fibrils [...]". This is not clear, and should be rephrased.

We reworded the sentence for clarity. The new text is: “These deposits show common structural and biochemical characteristics, including non-covalent self-complementary fibrils, resistance to proteolysis, insolubility in aqueous solution, binding of thioflavin dye, and generation of a cross-β X-ray diffraction pattern.”

3) Introduction: "Systemic light chain amyloidosis, frequently associated with multiple myeloma, was first described in 1848 when Dr. Bence Jones discovered a disease associated substance in urine [...]". Systemic AL amyloidosis is only occasionally associated with multiple myeloma and was not discovered first by Dr. Bence Jones (see Kyle. Amyloid 2011).

We rewrote the sentence and used the nomenclature recommended by Sipe et al., and added references to Kyle. Amyloid 2011 and Sipe et al. Amyloid 2014. The new text is: “Cases of systemic light chain amyloidosis (AL), associated with multiple myeloma, were first described during the Renaissance though the etiology of the disease was not clear. In 1848, Dr. Bence-Jones discovered a disease-associated substance in urine, later identified as immunoglobulin light chains (LC) (Kyle, 2001; Kyle, 2011; Dahlin and Dockerty, 1950; Glenner et al., 1970; Glenner et al., 1971; Glenner, 1973; Sipe et al., 2014).”

We added the reference to Kyle as a historical overview and kept the reference to Bence-Jones as he identified the substance in urine as protein.

To clarify that light-chain amyloidosis is only occasionally associated with multiple myeloma, we removed the word “frequently.” Also, we polished the paragraph due to the changes and the modified sentences are: “Cases of systemic light chain amyloidosis (AL), associated with multiple myeloma, were first described in the mid-19th century; however, the etiology of the disease was not clear. In 1848, Dr. Bence-Jones discovered the disease-associated substance in urine, though it was only later identified as immunoglobulin light chains (LC) associated with amyloidosis (Kyle, 2001; Kyle, 2011; Dahlin and Dockerty, 1950; Glenner et al., 1970; Glenner et al., 1971; Glenner, 1973; Sipe et al., 2014).”

4) Introduction: "This disease is characterized by overexpression of monoclonal LCs, which are able to form homo dimers termed Bence Jones proteins [...]". The use of Bence Jones protein referred to homo dimers of monoclonal light chain is inappropriate.

We removed the term Bence-Jones proteins. The new text is: “Systemic AL amyloidosis is characterized by overexpression of monoclonal LCs, which are able to form homo-dimers.”

5) Introduction: "LCs are produced by white blood cells [...]" is too generic. Use Plasma cells instead of white blood cells.

We accepted the reviewer’s suggestion. The text now reads: “LCs are produced by plasma cells and their final amino acid sequence is determined by somatic recombination (Sakano et al., 1979 and Marchalonis and Schluter, 1989).”

6) Introduction: "The mechanism of amyloid formation also suggests that shifting the equilibrium away from the amyloid prone monomer by stabilizing the dimer would hinder formation of amyloid fibrils (Figure 2). (Bulawa et al., 2012)." Quote also Bellotti et al., J Struct Biol 2000.

Belloti et al. J Struc Biol 2000 reference added.

7) Introduction: "The monomer dimer equilibrium of VLs suggests that systemic LC amyloidosis [...]". The use of systemic AL amyloidosis, according to international guidelines on nomenclature, would be preferable (see Sipe et al., Amyloid 2014).

We changed the nomenclature to that recommended by Sipe et al., Amyloid 2014. The reference was added earlier in the Introduction. New text is: “The monomer-dimer equilibrium of VLs suggests that systemic AL amyloidosis may be mitigated by binding ligands to the cavity at the VL dimer interface (Figure 2).”

8) Materials and methods: The addition of a short paragraph on the in silico search performed in the Drugbank website, including which type of search (ChemQuery? Other?), searching options and applied filters, would be useful.

We expanded our explanation of how we used the DrugBank service. Since we did not want to apply too many filters on potential results, we only used a single search parameter. The new text is: “Amyloid formation was monitored with Thioflavin T assays and electron microscopy analysis, and we used molecules that inhibited amyloid fibril formation at concentrations below 1 mM to search DrugBank to identify biomedical compounds with a similar structure that would potentially inhibit the formation of amyloid fibrils (Edmundson et al., 1976). We selected structures in DrugBank with a similarity threshold of 0.3. Search filters were not applied to any other parameters. We dissolved the identified molecules in either water or 20% DMSO for subsequent use in assays of amyloid formation.”

9) Figure 4: Replace 0.5 μM with 0.5 μm in the scale bar description. Define FI in the legend. Add units of measure in all y axes of panels A, B and C.

We replaced the units in the legend, defined Fl as fluorescence, and added units on y-axes on the graph.

10) Figure 5: Define [B], [L] and [B]/[U] in the legend. Add units of measure in all x and y axes of panels A and B.

We added units of measure in the legend. New sentence is: “[B], [L], and [U] are bound, total, and unbound concentrations of ligand in μM.”

11) Figure 7: Add units of measure in all y axes of panels A to F.

Added units at y axes.

12) Discussion: "The most significant outcome is the complete absence of soluble monomer in complex with the ligand, which verifies our conjecture that the binding of ligands to VLs induces dimerization". Does binding of ligands to VLs induce dimerization or do ligands bind and stabilize dimers?

We reworded this sentence to better describe the effect of ligand binding. The new text is: “The most significant outcome is the complete absence of soluble monomer in complex with the ligand, which verifies our conjecture that the ligands bind and stabilize dimers.”

13) Discussion: "While the light chain amino acid sequence differs between patients, the characteristic hydrophobic cavity of the VL homo dimer formed by overexpressed LCs is conserved". This point is central to the more broaden applicability of the therapeutic approach suggested in this study. This point should be better explained and appropriate references should be quoted to circumstantiate this claim.

We added an explanation to the last paragraph of the Discussion. The new text is: “The ability of ligands to hinder the aggregation of VLs into amyloid fibrils by stabilizing VL dimers indicates a promising approach for treatment of systemic light chain amyloidosis disease. […] Although our experiments were performed using a single VL variant, LCs of both λ and κ maintain a similar tertiary structure and hydrophobic cavity (Qin et al., 2007; Wetzel, 1994; Brumshtein et al., 2014; Bellotti, Mangione, and Merlini, 2000; Cohn and Edsall, 1943; Laue et al., 1992).”

https://doi.org/10.7554/eLife.10935.014

Article and author information

Author details

  1. Boris Brumshtein

    1. Department of Biological Chemistry, Howard Hughes Medical Institute, UCLA, Los Angeles, United States
    2. UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, United States
    3. Department of Chemistry and Biochemistry, UCLA, Los Angeles, United States
    Contribution
    BB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents
    Contributed equally with
    Shannon R Esswein
    For correspondence
    boris@mbi.ucla.edu
    Competing interests
    The authors declare that no competing interests exist.
  2. Shannon R Esswein

    1. Department of Biological Chemistry, Howard Hughes Medical Institute, UCLA, Los Angeles, United States
    2. UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, United States
    3. Department of Chemistry and Biochemistry, UCLA, Los Angeles, United States
    Contribution
    SRE, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents
    Contributed equally with
    Boris Brumshtein
    For correspondence
    sesswein@mbi.ucla.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0002-5142-0190
  3. Lukasz Salwinski

    1. Department of Biological Chemistry, Howard Hughes Medical Institute, UCLA, Los Angeles, United States
    2. UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, United States
    3. Department of Chemistry and Biochemistry, UCLA, Los Angeles, United States
    Contribution
    LS, Conception and design, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  4. Martin L Phillips

    1. UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, United States
    2. Department of Chemistry and Biochemistry, UCLA, Los Angeles, United States
    Contribution
    MLP, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents
    Competing interests
    The authors declare that no competing interests exist.
  5. Alan T Ly

    1. Department of Biological Chemistry, Howard Hughes Medical Institute, UCLA, Los Angeles, United States
    2. UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, United States
    3. Department of Chemistry and Biochemistry, UCLA, Los Angeles, United States
    Contribution
    ATL, Acquisition of data, Drafting or revising the article, Contributed unpublished essential data or reagents
    Competing interests
    The authors declare that no competing interests exist.
  6. Duilio Cascio

    1. Department of Biological Chemistry, Howard Hughes Medical Institute, UCLA, Los Angeles, United States
    2. UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, United States
    3. Department of Chemistry and Biochemistry, UCLA, Los Angeles, United States
    Contribution
    DC, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents
    Competing interests
    The authors declare that no competing interests exist.
  7. Michael R Sawaya

    1. Department of Biological Chemistry, Howard Hughes Medical Institute, UCLA, Los Angeles, United States
    2. UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, United States
    3. Department of Chemistry and Biochemistry, UCLA, Los Angeles, United States
    Contribution
    MRS, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents
    Competing interests
    The authors declare that no competing interests exist.
  8. David S Eisenberg

    1. Department of Biological Chemistry, Howard Hughes Medical Institute, UCLA, Los Angeles, United States
    2. UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, United States
    3. Department of Chemistry and Biochemistry, UCLA, Los Angeles, United States
    Contribution
    DSE, Conception and design, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    david@mbi.ucla.edu
    Competing interests
    The authors declare that no competing interests exist.

Funding

National Institutes of Health (AG048120)

  • Boris Brumshtein
  • Shannon R Esswein
  • Lukasz Salwinski
  • Martin L Phillips
  • Alan T Ly
  • Duilio Cascio
  • Michael R Sawaya
  • David S Eisenberg

National Institute of General Medical Sciences (R25GM055052)

  • Alan T Ly

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Mike Collazo for crystallization screens at the UCLA-DOE crystallization facility, M Capel, K Rajashankar, N Sukumar, J Schuermann, I Kourinov, and F Murphy for facilitating X-ray data collection experiments at the NE-CAT beamline 24-ID-C of APS, and Dr. Julian Whitelegge and Dr. Meytal Landau for discussions. We thank HHMI, NIH AG 04812 and NIGMS R25GM055052 for support. We thank Dr. Jeffery W Kelly and other eLife reviewers for their comments that helped us to improve this manuscript.

Reviewing Editor

  1. Jeffery W Kelly, Reviewing Editor, Scripps Research Institute, United States

Publication history

  1. Received: August 18, 2015
  2. Accepted: November 17, 2015
  3. Accepted Manuscript published: November 18, 2015 (version 1)
  4. Version of Record published: February 5, 2016 (version 2)

Copyright

© 2015, Brumshtein et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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