Co-aggregation and secondary nucleation in the life cycle of human prolactin/galanin functional amyloids

Synergistic-aggregation and cross-seeding by two different amyloid proteins/peptides are well evident in various neurological disorders. However, this phenomenon is not well studied in functional amyloid aggregation. Here, we show Prolactin (PRL) is associated with lactation in mammals and neuropeptide galanin (GAL), which are co-stored in the lactotrophs facilitates the synergic aggregation in the absence of secretory granules helper molecules glycosaminoglycans (GAGS). Interestingly, although each partner possesses homotypic seeding ability, a unidirectional cross-seeding of GAL aggregation can be mediated by PRL seeds. The specificity of co-aggregation by PRL and GAL along with unidirectional cross-seeding suggests tight regulation of functional amyloid formation during co-storage of these hormones in secretory granule biogenesis of female rat lactotrophs. Further mixed fibrils release the constituent functional hormone much faster than the corresponding individual amyloid formed in presence of GAGs, suggesting that co-aggregation of functionally distant hormones might have evolved for efficient storage, synergistic and rapid release of both hormones upon stimulation. The co-aggregation and cross seeding by two different hormones of completely different structures and sequences (PRL and GAL) suggest a novel mechanism of heterologous amyloid formation both in disease and functional amyloids.

Influence of The Expression Vector And of Its Elements On Recombinant hPRL Bacterial Synthesis: The Co-Directional Orientation of Replication And Transcription is Highly Critical

The aim of the present work was to define a bacterial expression system that is particularly efficient for the synthesis of human prolactin (hPRL). In previous work, the synthesis of rec-hPRL by the p1813-hPRL vector in E. coli HB2151 was >500 mg/L, while it was much lower now (2.5-4-fold), in the strains RB791 and RRI. The highest positive influence on rec-hPRL synthesis was due to the transcription-replication co-orientation of hPRL cDNA and the ori/antibiotic resistance gene, responsible for up to a ~5-6-fold higher expression yield. In conclusion, this work confirmed that each bacterial strain of E. coli has a genetic set that can allow a different level of heterologous protein synthesis. The individual study of each element indicated that its action critically depends on the reading orientation in which it is located inside the vector: co-directional orientation of replication and transcription, in fact, greatly increased the level of rec-hPRL expression.

Periplasmic synthesis and purification of the human prolactin antagonist Δ1-11-G129R-hPRL

The human prolactin antagonist Δ1-11-G129R-hPRL is a 21.9 kDa recombinant protein with 188 amino acids that downregulates the proliferation of a variety of cells expressing prolactin receptors. Periplasmic expression of recombinant proteins in E. coli has been considered an option for obtaining a soluble and correctly folded protein, as an alternative to cytoplasmic production. The aim of this work was, therefore, to synthesize for the first time, the Δ1-11-G129R-hPRL antagonist, testing different activation temperatures and purifying it by classical chromatographic techniques. E. coli BL21(DE3) strain was transformed with a plasmid based on the pET25b( +) vector, DsbA signal sequence and the antagonist cDNA sequence. Different doses of IPTG were added, activating under different temperatures, and extracting the periplasmic fluid via osmotic shock. The best conditions were achieved by activating at 35 °C for 5 h using 0.4 mM IPTG, which gave a specific expression of 0.157 ± 0.015 μg/mL/A600 at a final optical density of 3.43 ± 0.13 A600. Purification was carried out by nickel-affinity chromatography followed by size-exclusion chromatography, quantification being performed via high-performance size-exclusion chromatography (HPSEC). The prolactin antagonist was characterized by SDS-PAGE, Western blotting, reversed-phase high-performance liquid chromatography (RP-HPLC) and MALDI-TOF–MS. The final product presented > 95% purity and its antagonistic effects were evaluated in vitro in view of potential clinical applications, including inhibition of the proliferation of cancer cells overexpressing the prolactin receptor and specific antidiabetic properties, taking also advantage of the fact that this antagonist was obtained in a soluble and correctly folded form and without an initial methionine.

Periplasmic Synthesis and Purification of the Human Prolactin Antagonist Δ1-11-G129R-hPRL

The human prolactin antagonist Δ1-11-G129R-hPRL is a 21.9 kDa recombinant protein with 188 amino acids that downregulates the proliferation of a variety of cells expressing prolactin receptors. Periplasmic expression of recombinant proteins in E. coli has been considered an option for obtaining a soluble and correctly folded protein, as an alternative to cytoplasmic production. The aim of this work was, therefore, to synthesize for the first time, the Δ1-11-G129R-hPRL antagonist, testing different activation temperatures and purifying it by classical chromatographic techniques. E. coli BL21(DE3) strain was transformed with a plasmid based on the pET25b(+) vector, DsbA signal sequence and the antagonist cDNA sequence. Different doses of IPTG were added, activating under different temperatures, and extracting the periplasmic fluid via osmotic shock. The best conditions were achieved by activating at 35 °C for 5 h using 0.4 mM IPTG, which gave a specific expression of 0.157±0.015 μg/mL/A600 at a final optical density of 3.43±0.13 A600. Purification was carried out by nickel-affinity chromatography followed by size-exclusion chromatography, quantification being performed via high-performance size-exclusion chromatography (HPSEC). The prolactin antagonist was characterized by SDS-PAGE, Western blotting, reversed-phase high-performance liquid chromatography (RP-HPLC) and MALDI-TOF-MS. The final product presented >95% purity and its antagonistic effects were evaluated in vitro in view of potential clinical applications, including inhibition of the proliferation of cancer cells overexpressing the prolactin receptor and specific antidiabetic properties, taking also advantage of the fact that this antagonist was obtained in a soluble and correctly folded form and without an initial methionine.

Transcription factor Pit-1 affects transcriptional timing in the dual-promoter human prolactin gene

Gene transcription occurs in short bursts interspersed with silent periods, and these kinetics can be altered by promoter structure. The effect of alternate promoter architecture on transcription bursting is not known. We studied the human prolactin (hPRL) gene that contains two promoters, a pituitary-specific promoter that requires the transcription factor Pit-1, and displays dramatic transcriptional bursting activity, and an alternate upstream promoter that is active in non-pituitary tissues. We studied large hPRL genomic fragments with luciferase reporters, and used bacterial artificial chromosome (BAC) recombineering to manipulate critical promoter regions. Stochastic switch mathematical modelling of single-cell time-lapse luminescence image data revealed that the Pit-1-dependent promoter showed longer, higher-amplitude transcriptional bursts. Knockdown studies confirmed that the presence of Pit-1 stabilised and prolonged periods of active transcription. Pit-1 therefore plays an active role in establishing the timing of transcription cycles, in addition to its cell-specific functions.

KORELASI KADAR PROLAKTIN DAN OKSITOSIN DENGAN LAMA AMENORE LAKTASI PADA IBU MENYUSUI EKSKLUSIF

Prolaktin dan oksitosin merupakan hormon yang berperan penting dalam proses laktasi. Penelitian ini bertujuan untuk mengetahui korelasi kadar prolaktin dan oksitosin dengan lama amenore laktasi pada ibu menyusui eksklusif. Penelitian ini mneggunakan desain cross sectional, observasional terhadap 48 ibu menyusui eksklusif di wilayah kerja Puskesmas Belimbing Padang, September 2015 – Juni 2016. Sampel dipilih berdasarkan cluster random sampling. Pemeriksaan kadar prolaktin dan oksitosin dilakukan dilaboratorium Biomedik menggunakan Human Prolactin ELISA Kit dan Human Oxytocin ELISA Kit. Korelasi kadar prolaktin dan oksitosin dengan lama amenore laktasi diuji dengan menggunakan uji korelasi Spearman. Hasil penelitian menunjukkan terdapat korelasi positif yang lemah dan signifikan antara kadar prolaktin dengan lama amenore laktasi (r=0,331; p=0,022), dan terdapat korelasi negatif yang sangat lemah dan tidak signifikan antara kadar oksitosin dengan lama amenore laktasi (r=-0,085; p=0,565). Kesimpulan, semakin tinggi kadar prolaktin maka semakin bertambah lama amenore laktasi dan semakin tinggi kadar oksitosin maka semakin berkurang lama amenore laktasi.