The global cell culture market is experiencing significant growth due to the rapid advancement in antibody-based and cell-based therapies. Both rely on the capacity of different living factories, namely prokaryotic and eukaryotic cells, plants or animals for reliable and mass production. The ability to improve production yield is of important concern. Among many strategies pursued, optimizing the complex nutritional requirements for cell growth and protein production has been frequently performed via culture media component titration and serum replacement. The addition of specific ingredients into culture media to modulate host cells' metabolism has also recently been explored. In this study, we examined the use of extracted bioactive components of the microalgae Chlorella vulgaris, termed chlorella growth factor (CGF), as a cell culture additive for serum replacement and protein expression induction. We first established a chemical fingerprint of CGF using ultraviolet-visible spectroscopy and liquid chromatography-mass spectrometry and evaluated its ability to enhance cell proliferation in mammalian host cells. CGF successfully promoted the growth of Chinese hamster ovary (CHO) and mesenchymal stem cells (MSC), in both 2D and 3D cell cultures under reduced serum conditions for up to 21 days. In addition, CGF preserved cell functions as evident by an increase in protein expression in CHO cells and the maintenance of stem cell phenotype in MSC. Taken together, our results suggest that CGF is a viable culture media additive and growth matrix component, with wide ranging applications in biotechnology and tissue engineering.
Osteogenic transcription factor Runx2 is essential for osteoblast differentiation. The activity of Runx2 is tightly regulated at transcriptional as well as post-translational level. However, regulation of Runx2 stability by ubiquitin mediated proteasomal degradation by E3 ubiquitin ligases is little-known. Here, for the first time we demonstrate that Skp2, an SCF family E3 ubiquitin ligase negatively targets Runx2 by promoting its polyubiquitination and proteasome dependent degradation. Co-immunoprecipitation studies revealed that Skp2 physically interacts with Runx2 both in a heterologous as well as physiologically relevant system. Functional consequences of Runx2-Skp2 physical interaction were then assessed by promoter reporter assay. We show that Skp2-mediated downregulation of Runx2 led to reduced Runx2 transactivation and osteoblast differentiation. On the contrary, inhibition of Skp2 restored Runx2 levels and promoted osteoblast differentiation. We further show that Skp2 and Runx2 proteins are co-expressed and show inverse relation in vivo such as in lactating, ovariectomized and estrogen-treated ovariectomized animals. Together, these data demonstrate that Skp2 targets Runx2 for ubiquitin mediated degradation and hence negatively regulate osteogenesis. Therefore, the present study provides a plausible therapeutic target for osteoporosis or cleidocranial dysplasia caused by the heterozygous mutation of Runx2 gene.
Here we report a photoactive supramolecular assembly that is multifunctional and constructed by covalently linking four receptor molecules (cucurbit[7]uril) to a porphyrin derivative with suitable linkers. While this molecular platform serves very efficiently as a light-triggered broad-spectrum antibacterial agent, owing to its negligible dark cytotoxicity and the presence of host molecules (CB7), it can also be utilized as a vehicle to carry drug molecules for a combined chemo and photodynamic cancer therapy.
Perturbed stability of regulatory proteins is a major cause of transformations leading to cancer, including several leukemia subtypes. Here, for the first time we demonstrate that E6-associated protein (E6AP), an E3 ubiquitin ligase negatively targets MAX binding protein MNT for ubiquitin-mediated proteasome degradation and impedes ATRA mediated myeloid cell differentiation. MNT is a member of the Myc/Max/Mad network of transcription factor that regulates cell proliferation, differentiation, cellular transformation and tumorigenesis. Wild-type E6AP promoted proteasome dependent degradation of MNT, while catalytically inactive E6AP having cysteine replaced with alanine at amino-acid 843 position (E6APC843A) rather stabilized it. Further, these proteins physically associated with each other both in non-myeloid (HEK293T) and myeloid cells. MNT overexpression induced G0-G1 growth arrest and promoted myeloid differentiation while its knockdown mitigated even ATRA induced differentiation suggesting MNT to be crucial for myeloid differentiation. We further showed that ATRA inhibited E6AP and stabilized MNT expression by protecting it from E6AP mediated ubiquitin-proteasome degradation. Notably, E6AP knockdown in HL60 cells restored MNT expression and promoted myeloid differentiation. Taken together, our data demonstrated that E6AP negatively regulates granulocytic differentiation by targeting MNT for degradation which is required for growth arrest and subsequent myeloid differentiation by various differentiation inducing agents.
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