Mammalian cells as biopharmaceutical production hosts in the age of omics

Mammalian cells are important hosts for the production of a wide range of biopharmaceuticals due to their ability to produce correctly folded and glycosylated proteins. Compared to microbes and yeast, however, the productivity of mammalian cells is low because of their comparatively slow growth rate, tendency to undergo apoptosis, and low production capacities. While much effort has been invested in the engineering of mammalian cells with superior production characteristics, the success of these approaches has been limited to date. One factor responsible for this lack of success is our limited understanding of the cellular basis for high productivity, and of how discrete mechanisms within a cell contribute to the overall phenotype. Aiming to measure and characterize all cellular components at different functional levels, omics technologies have the potential to improve our understanding of mammalian cell physiology, elucidating new targets for the generation of a superior host cell line. This review provides a comprehensive analysis of recent examples of omics studies in the context of mammalian cells as production hosts, highlighting both the challenges and successes in the application of these powerful technologies.     

Activation of recA protein. The open helix model for LexA cleavage.

RecA protein is induced by the binding of DNA and ATP to become active in the hydrolysis of ATP and the cleavage of repressors. These reactions appear to depend on the structural state of the protein polymerized along the DNA, i.e. a helical coat of six RecA per turn of 95 to 100 A pitch. In support of this model of the active conformation, it was shown that high concentrations of salt also induce this helical polymerized state as well as the enzymatic activities. Here, we describe that, in vitro and with the non-hydrolyzable analogue ATP gamma S, RNA and heparin can also induce both the structural transition and the enzymatic activation of RecA to LexA cleavage in accordance with the model. RNA and heparin do not support the reaction in the presence of ATP, and they do not induce the hydrolysis of ATP either, suggesting that, in contrast to ATP gamma S, the nucleotide is not bound stably enough, and that the combined affinities of polynucleotide and ATP actually modulate the discrimination of RecA for the various possible inducers in vivo.     

AtMND1 is required for homologous pairing during meiosis in Arabidopsis

Background  

Pairing of homologous chromosomes at meiosis is an important requirement for recombination and balanced chromosome segregation among the products of meiotic division. Recombination is initiated by double strand breaks (DSBs) made by Spo11 followed by interaction of DSB sites with a homologous chromosome. This interaction requires the strand exchange proteins Rad51 and Dmc1 that bind to single stranded regions created by resection of ends at the site of DSBs and promote interactions with uncut DNA on the homologous partner. Recombination is also considered to be dependent on factors that stabilize interactions between homologous chromosomes. In budding yeast Hop2 and Mnd1 act as a complex to promote homologous pairing and recombination in conjunction with Rad51 and Dmc1.     

Complexes of RecA protein in solution. A study by small angle neutron scattering

RecA complexes on DNA and self-polymers were analysed by small-angle neutron scattering in solution. By Guinier analysis at small angles and by model analysis of a subsidiary peak at wider angles, we find that the filaments fall into two groups: the DNA complex in the presence of ATP gamma S, an open helix with pitch 95 A, a cross-sectional radius of gyration of 33 A and a mass per length of about six RecA units per turn, which corresponds to the state of active enzyme; and the compact form (bound to single-stranded DNA in the absence of ATP, or binding ATP gamma S in the absence of DNA, or just the protein on its own), a helical structure with pitch 70 A, cross-sectional radius of gyration 40 A and mass per length about five RecA units per turn, which corresponds to the conditions of inactive enzyme. The results are discussed in the perspective of unifying previous conflicting structural results obtained by electron microscopy.     

Status and Conservation of Douc Langurs (Pygathrix nemaeus) in Laos

A recent systematic survey program of protected areas in Laos documented douc langurs (Pygathrix nemaeus) widely between 14°25N and 18°38N. Their precise northern limit is unclear as security constraints prevented survey there, but the species is unlikely to occur in Laos north of 20°. All our records are of the nominate, red-shanked form, P. n. nemaeus, though in the far south, greatly reduced red on the legs suggests intergradation with another form. Our records are overwhelmingly from evergreen or semievergreen forest and from a wide altitudinal range (200–1600 m). In most sites habitat was little degraded, but whether this reflects choice for such habitats, or the generally lower hunting pressure within them, is unclear. The largest population occurs over 3000 km ² of the Nam Theun basin and surroundings, encompassing the protected areas of Nakai-Nam Theun, Hin Namno and the proposed Nam Theun Extension. This is the most important population of red-shanked langurs in the world. Laos supports globally the vast majority of their conservable population. Douc langurs are threatened in Laos by habitat fragmentation increasing their vulnerability to the existing high hunting levels. They are a favored target for local consumption and for local and Vietnamese hunters and traders. Protective measures should center around implementing habitat conservation and antihunting measures in the recently-declared National Biodiversity Conservation Areas. A recent government initiative to reduce gun use in rural areas is likely to have a strong positive effect on the population. The best protection for some time will remain the inaccessibility of their habitat. Accordingly, road-building into remote forest and piecemeal clearance at its edges should thus be minimized. Cross-border attention should be given to wildlife trading, including measures to reduce market demand.     

Detergent organisation in crystals of monomeric outer membrane phospholipase A

The structure of the detergent in crystals of outer membrane phospholipase A (OMPLA) has been determined using neutron diffraction contrast variation. Large crystals were soaked in stabilising solutions, each containing a different H(2)O/D(2)O contrast. From the neutron diffraction at five contrasts, the 12 A resolution structure of the detergent micelle around the protein molecule was determined. The hydrophobic beta-barrel surfaces of the protein molecules are covered by rings of detergent. These detergent belts are fused to neighbouring detergent rings forming a continuous three-dimensional network throughout the crystal. The thickness of the detergent layer around the protein varies from 7-20 A. The enzyme's active site is positioned just outside the hydrophobic detergent zone and is thus in a proper location to catalyse the hydrolysis of phospholipids in a natural membrane. Although the dimerisation face of OMPLA is covered with detergent, the detergent density is weak near the exposed polar patch, suggesting that burying this patch in the enzyme's dimer interface may be energetically favourable. Furthermore, these results indicate a crucial role for detergent coalescence during crystal formation and contribute to the understanding of membrane protein crystallisation.     

Small-angle neutron scattering with contrast variation reveals spatial relationships between the three subunits in the ternary cardiac troponin complex and the effects of troponin I phosphorylation

Small-angle neutron scattering with contrast variation has been used to determine the shapes and dispositions of the three subunits of cardiac troponin and to study the influence of phosphorylation on the structure. Three contrast variation series were collected on three different isotopically labeled variants of the cTnC/cTnI/cTnT(198-298) complex, one of which contained deuterated and bisphosphorylated cTnI. Analysis of the scattering data shows cTnT(198-298) interacting with a single lobe of a somewhat compacted cTnC that sits at one end of an elongated rodlike cTnI, covering about one-third of its length. The cTnT(198-298) sits near the center of the long cTnI axis. The components undergo significant conformational changes and reorientations in response to protein kinase A phosphorylation of cTnI. The rodlike cTnI bends sharply at the end interacting with the cTnC/cTnT(198-298) component, which reorients so as to maintain its contacts with cTnI while undergoing only a relatively small change in shape.     

Ebola virus matrix protein VP40 interaction with human cellular factors Tsg101 and Nedd4

The Ebola virus matrix protein VP40 is a major viral structural protein and plays a central role in virus assembly and budding at the plasma membrane of infected cells. For efficient budding, a full amino terminus of VP40 is required, which includes a PPXY and a PT/SAP motif, both of which have been proposed to interact with cellular proteins. Here, we report that Ebola VP40 can interact with cellular factors human Nedd4 and Tsg101 in vitro. We show that WW domain 3 of human Nedd4 is necessary and sufficient for binding to the PPXY motif of VP40, which requires an oligomeric conformation of VP40. Single particle electron microscopy reconstructions indicate that WW3 of Nedd4 is in close contact with the N-terminal domain of hexameric VP40. In contrast, the ubiquitin enzyme variant domain of Tsg101 was sufficient for binding to the PT/SAP motif of VP40, regardless of the oligomeric state of the matrix protein. These results suggest that hNedd4 and Tsg101 may play complimentary roles at a late stage of the assembly process, by recruiting cellular factors of two independent pathways to the site of budding at the plasma membrane.