Fig 1: Seeding and cross-seeding of PRL and GAL.(a) Schematic showing possible homo and hetero seeding with fibril elongation and surface-mediated secondary nucleation mechanism for seed-mediated fibril growth. (b–c) Homo-seeding of PRL and GAL. (Left panel) PRL and GAL homo-seeding. Normalized ThT fluorescence intensity values with time indicating aggregation of PRL/GAL and in the presence of different concentrations of PRL seeds and GAL seeds respectively (2% and 5% v/v). Only seeds and only PRL/GAL was used as controls. (Right panel) The corresponding EM images of PRL/GAL seeds alone and PRL/GAL monomer in presence of 5% PRL/GAL seeds showing fibrils formation by PRL/GAL homo-seeding. (d) Cross-seeding of PRL and GAL. (Left panel) Normalized ThT fluorescence intensity values with time indicating aggregation of GAL in the presence of different concentrations of PRL seeds (1%, 2%, and 5% v/v). However, PRL in presence of different percentages of GAL seeds does not show any aggregation (Right panel). Only seed and only GAL/PRL were used as controls where no aggregation is observed. (e) The lag times of GAL aggregation in presence of 2% and 5% (v/v) PRL seeds and GAL seeds are compared. The values represent mean ± SEM. The significance (***p = 0.001) is calculated using one-way ANOVA followed by an SNK post hoc test with a 95% confidence interval. (f) TEM images of GAL fibrils formed in presence of PRL seeds are shown. GAL fibrils formed in presence of 5% (v/v) PRL seeds are analyzed for frequency distribution (red arrows indicating the diameter of the fibrils measured for analysis). (g) (Left panel) Normalized frequency distribution of fibril diameter showing GAL fibrils formed in presence of PRL seeds have a similar diameter to GAL-Hep fibrils. A total of 200 random data points from different individual fibrils were collected from n = 3 independent experiments for the frequency distribution analysis. (Right panel) Average values of different fibril diameters are shown. Values represent mean ± SD. The statistical significance (***p = 0.001, **p = 0.01) is calculated by one-way ANOVA followed by an SNK post hoc test with a 95% confidence interval. (h) FTIR spectra showing fibrils of GAL +5% GAL seed and GAL +5% PRL seed are of similar secondary structure. Figure 3—source data 1.Seeding and cross-seeding of PRL and GAL.
Fig 2: Specific interaction drives co-aggregation of PRL and GAL.(a) Comparative ThT fluorescence showing amyloid formation by different pairs of hormones at days 0 and 15. PRL-CSA, GAL-Hep, PRL-GAL showed the highest ThT fluorescence signals after 15 days of incubation. Values represent mean ± SEM for n = 3 independent experiments. The statistical significance is calculated between day 0 and day 15 for each sample using a t-test. (b) The morphology observed under TEM for various hormones and the mixture of hormone samples is shown (after 15 days of incubation). Amorphous structures are seen for PRL-ACTH and GAL-GH; whereas PRL-GAL, PRL-CSA, GAL-Hep showed fibrillar morphology similar to amyloids. The experiment is performed three times with similar observations. (c) (Left panel) Surface Plasmon Resonance (SPR) spectra showing strong binding of PRL on immobilized GAL compared to other pairs of hormones. (Right panel) The dissociation constant (KD) of PRL to GAL showing strong interaction between PRL and GAL for their co-aggregation and co-storage. The experiments are performed three times with similar results. (d) Double immunofluorescence microscopic images of the anterior pituitary of female rats showing ACTH (red) and GH (green) expressing cells. The merged microscopic image (right) shows no co-localization of ACTH (red) and GH (green). The data indicate that ACTH and GH are not co-stored in the female rat anterior pituitary. The experiments were performed three times with similar observations. (e) Snapshot from in silico analysis (MD simulation) of PRL-GAL complex 1 using GROMOS 53a6 force field (when GAL is docked near residues 18–28 of PRL). (f) Snapshot showing MD simulation of PRL-GAL complex 2 (when GAL is docked near residues 80–88 of PRL) using GROMOS 53a6 force field. Complex 1 induced the formation of an antiparallel ß-sheet at the PRL-GAL interface (6–8 PRL and 24–26 GAL) and also an intra-molecular parallel ß-sheet in PRL itself (59–61 PRL and 149–151 PRL). Complex 2 shows the formation of a parallel ß-sheet constituted by the ß-strand from PRL and GAL (145–147 PRL and 4–6 GAL). (g) Snapshot of MD simulation of complex 2 using Amber ff99SB force field showing the appearance of parallel ß-sheet at 147–149 residue of PRL and 2–4 residue of GAL. The snap-shot of complex 1 is included in Figure 4—figure supplement 3. (h) A point mutation is introduced in the PRL of the complex 2 structure, which is Y147P to examine if there is a loss in the ß-sheet formation. The initial structure of the complex had the ß-sheet formed between residues 147–149 of PRL and residues 2–4 of GAL (Left panel), which went missing during the 400 ns MD simulation run of the mutated system (right panel). Figure 4—source data 1.Specific interaction drives co-aggregation of PRL and GAL. Figure 4—source data 2.Parameter values for surface plasmon resonance spectroscopy (SPR) table. Figure 4—source data 3.Parameter values for surface plasmon resonance spectroscopy (SPR).
Fig 3: Amyloid propensity and co-storage of PRL and GAL.(a) Schematic showing amino acid sequence and secondary structures of PRL & GAL with different color codes. (Upper panel) PRL is 191 amino acids in length and contains a four-helix bundle (green). The short helix and loop regions are also represented between helix-1 and helix 2 (shown in green and blue colors, respectively). (Lower panel) GAL showing 30 residue peptide with no definite secondary structure (Evans and Shine, 1991; Bersani et al., 1991). (b) (Left panel) The three-dimensional structure (obtained in Pymol) (Teilum et al., 2005) of PRL showing its major helices and two tryptophan residues (shown in purple) (PDB ID: 1RW5). (Right panel) Natively unstructured conformation of GAL is also shown. (c) TANGO algorithm showing the aggregation-prone residues of PRL and GAL at pH 6.0 (SGs relevant pH). The residues 18–29 and 80–88 of PRL showing amyloid aggregation potential. However, TANGO analysis of GAL revealed no amyloid aggregation propensity. Immunofluorescence studies showing (d) colocalization of PRL (red) and GAL (green) in the female rat anterior pituitary. (e) (left panel) Colocalization of amyloid fibrils (OC, green) and PRL (red) and amyloid fibrils (OC, green) and GAL (red) (Right panel) in the anterior pituitary of female rat. The merged microscopic image showing colocalization (yellow). The experiments (d-e) are performed three times with similar observations. Figure 1—source data 1.TANGO ß-Aggregation propensity. Figure 1—source data 2.Protein/peptide hormone sequence used in this study.
Fig 4: PRL-GAL homo and hetero amyloid life cycle for SGs.PRL and GAL form the amyloid fibrils in the presence of specific glycosaminoglycans (CSA and Hep, respectively), which can be auto-catalytically amplified by their respective seeding with preformed fibrils. This seeding however does not require any glycosaminoglycans. PRL-GAL also synergistically co-aggregate to form hybrid amyloid fibrils, which are not capable of seeding either to PRL or GAL. These amyloid fibril species can together or individually reconstitute the SGs of PRL-GA storage, which can release functional PRL and GAL into the extracellular space.
Fig 5: Monomer release from PRL and GAL amyloid.(a) The kinetics of monomer release from various amyloids showing the continuous release of monomeric hormones. The experiment is performed three times with similar results. Values represent mean ± SEM for n = 3 independent experiments. (b) Saturation concentrations of different released monomers from fibrils along with the monomeric controls are shown. Values represent mean ± SEM for n = 3 independent experiments. (c) The secondary structure of released monomers showing their corresponding native secondary structures as confirmed by the CD. (d) Nb2 cell proliferation study showing biological activity of released PRL from either PRL-CSA or PRL-GAL fibrils. Freshly dissolved protein was used as a control. Values represent mean ± SEM for n = 3 independent experiments. (e) EC50 values showing the released PRL monomers have similar bioactivity compared to freshly dissolved monomeric PRL. Values represent mean ± SEM for n = 3 independent experiments. Figure 5—source data 1.Monomer release from PRL and GAL amyloid.
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