Many protein-based biotherapeutics are produced in cultured Chinese hamster ovary (CHO) cell lines. Recent reports have demonstrated that translation of recombinant mRNAs and global control of the translation machinery via mammalian target of rapamycin (mTOR) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mTOR complex 1 (mTORC1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relationship between mTORC1 signalling, cell growth and proliferation and recombinant protein yields from mammalian cells, and whether this master regulating signalling pathway can be manipulated to enhance cell biomass and recombinant protein production (rPP) are not well explored. We have investigated mTORC1 signalling and activity throughout batch culture of a panel of sister recombinant glutamine synthetase-CHO cell lines expressing different amounts of a model monoclonal IgG4, to evaluate the links between mTORC1 signalling and cell proliferation, autophagy, recombinant protein expression, global protein synthesis and mRNA translation initiation. We find that the expression of the mTORC1 substrate 4E-binding protein 1 (4E-BP1) fluctuates throughout the course of cell culture and, as expected, that the 4E-BP1 phosphorylation profiles change across the culture. Importantly, we find that the eIF4E/4E-BP1 stoichiometry positively correlates with cell productivity. Furthermore, eIF4E amounts appear to be co-regulated with 4E-BP1 amounts. This may reflect a sensing of either change at the mRNA level as opposed to the protein level or the fact that the phosphorylation status, as well as the amount of 4E-BP1 present, is important in the co-regulation of eIF4E and 4E-BP1.
Human DSS1 associates with BRCA2, a tumour suppressor protein required for efficient recombinational DNA repair, but the biochemical function of DSS1 is not known. Orthologues of DSS1 are found in organisms such as budding yeast and fission yeast that do not have BRCA2-related proteins, indicating that DSS1 has a physiological role independent of BRCA2. The DSS1 orthologue in Saccharomyces cerevisiae has been shown to associate with the 26 S proteasome and, in the present paper, we report that in the distantly related fission yeast Schizosaccharomyces pombe, Dss1 associates with the 19 S RP (regulatory particle) of the 26 S proteasome. A role for S. pombe Dss1 in proteasome function is supported by three lines of evidence. First, overexpression of two components of the 19 S RP, namely Pad1/Rpn11 and Mts3/Rpn12, rescued the temperature-sensitive growth defect of the dss1 mutant. Secondly, the dss1 mutant showed phenotypes indicative of a defect in proteasome function: growth of the dss1 mutant was inhibited by low concentrations of L-canavanine, an amino acid analogue, and cells of the dss1 mutant accumulated high molecular mass poly-ubiquitylated proteins. Thirdly, synthetic growth defects were found when the dss1 mutation was combined with mutations in other proteasome subunit genes. These findings show that DSS1 has an evolutionarily conserved role as a regulator of proteasome function and suggest that DSS1 may provide a link between BRCA2 and ubiquitin-mediated proteolysis in human cells.
Alzheimer’s disease is the most common neurodegenerative disease, affecting ∼50% of humans by age 85. The disease process is associated with aggregation of the Aβ peptides, short 39–43 residue peptides generated through endoproteolytic cleavage of the Alzheimer’s precursor protein. While the process of aggregation of purified Aβ peptides in vitro is beginning to be well understood, little is known about this process in vivo. In the present study, we use the yeast Saccharomyces cerevisiae as a model system for studying Aβ-mediated aggregation in an organism in vivo. One ofthis yeast’s endogenous prions, Sup35/[PSI+], loses the ability to aggregate when the prion-forming domain of this protein is deleted. We show that insertion of Aβ peptide sequences in place of the original prion domain of this protein restores its ability to aggregate. However, the aggregates are qualitatively different from [PSI+] prions in their sensitivity to detergents and in their requirements on trans-acting factors that are normally needed for [PSI+] propagation. We conclude that we have established a useful new tool for studying the aggregation of Aβ peptides in an organism in vivo.
Cryptosporidium is a genus of single celled parasites capable of infecting a wide range of animals including humans. Cryptosporidium species are members of the phylum apicomplexa, which includes well-known genera such as Plasmodium and Toxoplasma. Cryptosporidium parasites cause a severe gastro-intestinal disease known as cryptosporidiosis. They are one of the most common causes of childhood diarrhoea worldwide, and infection can have prolonged detrimental effects on the development of children, but also can be life threatening to HIV/AIDS patients and transplant recipients. A variety of hosts can act as reservoirs, and Cryptosporidium can persist in the environment for prolonged times as oocysts. While there has been substantial interest in these parasites, there is very little progress in terms of treatment development and understanding the majority of the life cycle of this unusual organism. In this review, we will provide an overview on the existing knowledge of the biology of the parasite and the current progress in developing in vitro cultivation systems. We will then describe a synopsis of current and next generation approaches that could spearhead further research in combating the parasite.
Cryptosporidium is a protozoan, apicomplexan, parasite that poses significant risk to humans and animals, as a common cause of potentially fatal diarrhea in immunodeficient hosts. The parasites have evolved a number of unique biological features that allow them to thrive in a highly specialized parasitic lifestyle. For example, the genome of Cryptosporidium parvum is highly reduced, encoding only 3,805 proteins, which is also reflected in its reduced cellular and organellar content and functions. As such, its remnant mitochondrion, dubbed a mitosome, is one of the smallest mitochondria yet found. While numerous studies have attempted to discover the function(s) of the C. parvum mitosome, most of them have been focused on in silico predictions. Here, we have localized components of a biochemical pathway in the C. parvum mitosome, in our investigations into the functions of this peculiar mitochondrial organelle. We have shown that three proteins involved in the mitochondrial iron‐sulfur cluster biosynthetic pathway are localized in the organelle, and one of them can functionally replace its yeast homolog. Thus, it seems that the C. parvum mitosome is involved in iron‐sulfur cluster biosynthesis, supporting the organellar and cytosolic apoproteins. These results spearhead further research on elucidating the functions of the mitosome and broaden our understanding in the minimalistic adaptations of these organelles.
Cultured mammalian cells, particularly Chinese hamster ovary (CHO) cells, are widely exploited as hosts for the production of recombinant proteins, but often yields are limiting. Such limitations may be due in part to the misfolding and subsequent degradation of the heterologous proteins. Consequently we have determined whether transiently co-expressing yeast and/or mammalian chaperones that act to disaggregate proteins, in CHO cell lines, improve the levels of either a cytoplasmic (Fluc) or secreted (Gluc) form of luciferase or an immunoglobulin IgG4 molecule. Over-expression of the yeast 'protein disaggregase' Hsp104 in a CHO cell line increased the levels of Fluc more significantly than for Gluc although levels were not further elevated by over-expression of the yeast or mammalian Hsp70/40 chaperones. Over-expression of TorsinA, a mammalian protein related in sequence to yeast Hsp104, but located in the ER, significantly increased the level of secreted Gluc from CHO cells by 2.5-fold and to a lesser extent the secreted levels of a recombinant IgG4 molecule. These observations indicate that the over-expression of yeast Hsp104 in mammalian cells can improve recombinant protein yield and that over-expression of TorsinA in the ER can promote secretion of heterologous proteins from mammalian cells.
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