Folding of Truncated Granulin Peptides.

Granulins are a family of unique protein growth factors which are found in a range of species and have several bioactivities that include cell proliferation and wound healing. They typically contain six disulfide bonds, but the sequences, structures and bioactivities vary significantly. We have previously shown that an N-terminally truncated version of a granulin from the human liver fluke, Opisthorchis viverrini, can fold independently into a "mini-granulin" structure and has potent wound healing properties in vivo. The incorporation of a non-native third disulfide bond, with respect to the full-length granulin module, was critical for the formation of regular secondary structure in the liver fluke derived peptide. By contrast, this third disulfide bond is not required for a carp granulin-1 truncated peptide to fold independently. This distinction led us to explore granulins from the zebrafish model organism. Here we show that the mini-granulin fold occurs in a naturally occurring paragranulin (half-domain) from zebrafish, and is also present in a truncated form of a full-length zebrafish granulin, suggesting this structure might be a common property in either naturally occurring or engineered N-terminally truncated granulins and the carp granulin-1 folding is an anomaly. The in vitro folding yield is significantly higher in the naturally occurring paragranulin, but only the truncated zebrafish granulin peptide promoted the proliferation of fibroblasts consistent with a growth factor function, and therefore the function of the paragranulin remains unknown. These findings provide insight into the folding and evolution of granulin domains and might be useful in the elucidation of the structural features important for bioactivity to aid the design of more potent and stable analogues for the development of novel wound healing agents.

A novel granulin homologue isolated from the jellyfish Cyanea capillata promotes proliferation and migration of human umbilical vein endothelial cells through the ERK1/2-signaling pathway.

Jellyfish grow rapidly and have a strong regenerative ability, indicating that they may express high levels of growth factors. Therefore, the aim of this research was to isolate the growth-promoting components from the jellyfish Cyanea capillata (C. capillata) and to further explore the underlying mechanisms. In this study, we first isolated and identified a novel polypeptide from C. capillata tentacles using size-exclusion chromatography followed by reverse-phase HPLC. This peptide, consisting of 58 amino acids (MW 5782.9 Da), belonged to the granulin (GRN) family of growth factors; thus, we named it Cyanea capillata granulin-1 (CcGRN-1). Second, using CCK-8 assay and flow cytometry, we verified that CcGRN-1 at the 0.5 µg/ml concentration could promote cell proliferation and increase the expression of cell-cycle proteins (CyclinB1 and CyclinD1). Third, signaling pathways studies showed that CcGRN-1 could activate the PI3K/Akt- and ERK1/2 MAPK-signaling pathways but not the JNK MAPK- or NF-κB-signaling pathways. Subsequently, we further confirmed that the CcGRN-1-induced cell proliferation and migration were associated only with the ERK1/2 MAPK-signaling pathway. Considering all of these factors, CcGRN-1, as the first jellyfish-derived GRN homologue, possesses growth-promoting properties and may be a candidate for novel therapeutics to promote human wound healing in unfavorable conditions.

Granulin 1 Promotes Retinal Regeneration in Zebrafish.

Purpose: Retinal degenerative diseases can progress to severe reductions of vision. In general, the changes are permanent in higher vertebrates, including humans; however, retinal regeneration can occur in lower vertebrates, such as amphibians and teleost fish. Progranulin is a secreted growth factor that is involved in normal development and wound-healing processes. We have shown that progranulin promotes the proliferation of retinal precursor cells in mouse retinas. The purpose of this study was to investigate the role played by granulin 1 (grn1) in the retinal regeneration in zebrafish. Methods: We injured the retina of zebrafish with needle puncturing, and the retinas were examined at different times after the injury. We also checked the proliferation and the expression of retinal regeneration-related genes after knockdown of grn1 by electroporation with morpholino oligonucleotides (MO) and intravitreal injection of recombinant grn1. Results: Our results showed that the level of grn1 was highly increased after retinal injury, and it was expressed in various types of retinal cells. A knockdown of grn1 reduced the proliferation of Müller glial cells in zebrafish eyes undergoing retinal regeneration. The knockdown of grn1 also reduced the expression of achaete-scute homolog 1a (ascl1a), an important factor in retinal regeneration. An intravitreal injection of recombinant grn1 led to a proliferation of Müller glial cells and an increase in the expression of retinal regeneration-related genes, such as ascl1a and lin28. Conclusions: These findings suggested that grn1 should be considered as a target for stimulating the dedifferentiation of Müller glial cells and retinal regeneration.

Granulin A Synergizes with Cisplatin to Inhibit the Growth of Human Hepatocellular Carcinoma.

Cisplatin is one of the most potent chemotherapy drugs widely used for cancer treatment. However, due to resistance and toxicity, the application of cisplatin for the treatment of hepatocellular carcinoma (HCC) is limited. Our previous study has shown that granulin A (GRN A), an anticancer peptide, is able to interact with enolase1 (ENO1) and inhibit the growth of HCC in vitro. In the present study, we studied the synergistic effect of the combination of cisplatin and GRN A for the inhibitory effect on HCC. An 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and Chou-Talalay approaches revealed that the combination of GRN A and cisplatin displayed potent synergistic effect. The colony formation and cell viability of HCC cells were inhibited significantly in cells treated with the combination of cisplatin and GRN A, compared with cells treated with cisplatin or GRN A alone. Overexpression of ENO1 diminished the synergistic effect of GRN A and cisplatin in HCC cells. The combination of the two drugs exhibited a more obvious inhibitory effect on cancer cell apoptosis, as analyzed by the cytometry flow, mitochondrial membrane potential (MMP) and western blot analysis. An in vivo study confirmed that the combined use of the two drugs displayed more potent antitumor activity compared to mice treated with cisplatin and GRN A alone; the inhibitory rate of tumor growth was 65.46% and 68.94%, respectively, in mice treated with GRN A and cisplatin. However, the inhibitory rate increased to 86.63% in mice treated with the combination of the two drugs. This study provides evidence that the combination of GRN A and cisplatin is able to sensitize the liver cancer to cisplatin, and that targeting ENO1 is a promising approach for enhancing the antitumor activity of cisplatin.

Structural Variants of a Liver Fluke Derived Granulin Peptide Potently Stimulate Wound Healing.

Granulins are a family of growth factors involved in cell proliferation. The liver-fluke granulin, Ov-GRN-1, isolated from a carcinogenic liver fluke Opisthorchis viverrini, can significantly accelerate wound repair in vivo and in vitro. However, it is difficult to express Ov-GRN-1 in recombinant form at high yield, impeding its utility as a drug lead. Previously we reported that a truncated analogue ( Ov-GRN12-35_3s) promotes healing of cutaneous wounds in mice. NMR analysis of this analogue indicates the presence of multiple conformations, most likely as a result of proline cis/ trans isomerization. To further investigate whether the proline residues are involved in adopting the multiple confirmations, we have synthesized analogues involving mutation of the proline residues. We have shown that the proline residues have a significant influence on the structure, activity, and folding of Ov-GRN12-35_3s. These results provide insight into improving the oxidative folding yield and bioactivity of Ov-GRN12-35_3s and might facilitate the development of a novel wound healing agent.

Conotoxin Φ-MiXXVIIA from the Superfamily G2 Employs a Novel Cysteine Framework that Mimics Granulin and Displays Anti-Apoptotic Activity.

Conotoxins are a large family of disulfide-rich peptides that contain unique cysteine frameworks that target a broad range of ion channels and receptors. We recently discovered the 33-residue conotoxin Φ-MiXXVIIA from Conus miles with a novel cysteine framework comprising three consecutive cysteine residues and four disulfide bonds. Regioselective chemical synthesis helped decipher the disulfide bond connectivity and the structure of Φ-MiXXVIIA was determined by NMR spectroscopy. The 3D structure displays a unique topology containing two ß-hairpins that resemble the N-terminal domain of granulin. Similar to granulin, Φ-MiXXVIIA promotes cell proliferation (EC50 17.85 µm) while inhibiting apoptosis (EC50 2.2 µm). Additional framework XXVII sequences were discovered with homologous signal peptides that define the new conotoxin superfamily G2. The novel structure and biological activity of Φ-MiXXVIIA expands the repertoire of disulfide-rich conotoxins that recognize mammalian receptors.