Heterologous expression of the human potassium channel Kv2.1 in clonal mammalian cells by direct cytoplasmic microinjection of cRNA

Human protoporphyrinogen oxidase IX (hPPO) is an oxygen-dependent enzyme catalyzing the penultimate step in the heme biosynthesis pathway. Mutations in the enzyme are linked to variegate porphyria, an autosomal dominant metabolic disease. Here we investigated eukaryotic cells as alternative systems for heterologous expression of hPPO, as the use of a traditional bacterial-based system failed to produce several clinically relevant hPPO variants. Using bacterially-produced hPPO, we first analyzed the impact of N-terminal tags and various detergent on hPPO yield, and specific activity. Next, the established protocol was used to compare hPPO constructs heterologously expressed in mammalian HEK293T17 and insect Hi5 cells with prokaryotic overexpression. By attaching various fusion partners at the N- and C-termini of hPPO we also evaluated the influence of the size and positioning of fusion partners on expression levels, specific activity, and intracellular targeting of hPPO fusions in mammalian cells. Overall, our results suggest that while enzymatically active hPPO can be heterologously produced in eukaryotic systems, the limited availability of the intracellular FAD co-factor likely negatively influences yields of a correctly folded protein making thus the E.coli a system of choice for recombinant hPPO overproduction. At the same time, PPO overexpression in eukaryotic cells might be preferrable in cases when the effects of post-translational modifications (absent in bacteria) on target protein functions are studied.

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Interactions of GMP with Human Glrx3 and with Saccharomyces cerevisiae Grx3 and Grx4 Converge in the Regulation of the Gcn2 Pathway

Applied and Environmental Microbiology ◽

10.1128/aem.00221-20 ◽

2020 ◽

Vol 86 (14) ◽

Author(s):

Mónica A. Mechoud ◽

Nuria Pujol-Carrion ◽

Sandra Montella-Manuel ◽

Maria Angeles de la Torre-Ruiz

Keyword(s):

Saccharomyces Cerevisiae ◽

Heterologous Expression ◽

Mammalian Cells ◽

Iron Homeostasis ◽

Life Extension ◽

Iron Sulfur Clusters ◽

Content Type ◽

Functional Conservation ◽

Iron Sulfur ◽

Cell Life

ABSTRACT The human monothiol glutaredoxin Glrx3 (PICOT) is ubiquitously distributed in cytoplasm and nuclei in mammalian cells. Its overexpression has been associated with the development of several types of tumors, whereas its deficiency might cause retardation in embryogenesis. Its exact biological role has not been well resolved, although a function as a chaperone distributing iron/sulfur clusters is currently accepted. Yeast humanization and the use of a mouse library have allowed us to find a new partner for PICOT: the human GMP synthase (hGMPs). Both proteins carry out collaborative functions regarding the downregulation of the Saccharomyces cerevisiae Gcn2 pathway under conditions of nutritional stress. Glrx3/hGMPs interact through conserved residues that bridge iron/sulfur clusters and glutathione. This mechanism is also conserved in budding yeast, whose proteins Grx3/Grx4, along with GUA1 (S. cerevisiae GMPs), also downregulate the integrated stress response (ISR) pathway. The heterologous expression of Glrx3/hGMPs efficiently complements Grx3/Grx4. Moreover, the heterologous expression of Glrx3 efficiently complements the novel participation in chronological life span that has been characterized for both Grx3 and Grx4. Our results underscore that the Glrx3/Grx3/Grx4 family presents an evolutionary and functional conservation in signaling events that is partly related to GMP function and contributes to cell life extension. IMPORTANCE Saccharomyces cerevisiae is an optimal eukaryotic microbial model to study biological processes in higher organisms despite the divergence in evolution. The molecular function of yeast glutaredoxins Grx3 and Grx4 is enormously interesting, since both proteins are required to maintain correct iron homeostasis and an efficient response to oxidative stress. The human orthologous Glrx3 (PICOT) is involved in a number of human diseases, including cancer. Our research expanded its utility to human cells. Yeast has allowed the characterization of GMP synthase as a new interacting partner for Glrx3 and also for yeast Grx3 and Grx4, the complex monothiol glutaredoxins/GMPs that participate in the downregulation of the activity of the Gcn2 stress pathway. This mechanism is conserved in yeast and humans. Here, we also show that this family of glutaredoxins, Grx3/Grx4/Glrx3, also has a function related to life extension.

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TheNitrosopumilus maritimusCdvB, but Not FtsZ, Assembles into Polymers

Archaea ◽

10.1155/2013/104147 ◽

2013 ◽

Vol 2013 ◽

pp. 1-10 ◽

Cited By ~ 6

Author(s):

Kian-Hong Ng ◽

Vinayaka Srinivas ◽

Ramanujam Srinivasan ◽

Mohan Balasubramanian

Keyword(s):

Cell Division ◽

Heterologous Expression ◽

Fission Yeast ◽

Mammalian Cells ◽

Expression System ◽

Related Protein ◽

Heterologous Expression System

Euryarchaeota and Crenarchaeota are two major phyla of archaea which use distinct molecular apparatuses for cell division. Euryarchaea make use of the tubulin-related protein FtsZ, while Crenarchaea, which appear to lack functional FtsZ, employ the Cdv (cell division) components to divide. Ammonia oxidizing archaeon (AOA)Nitrosopumilus maritimusbelongs to another archaeal phylum, the Thaumarchaeota, which has both FtsZ and Cdv genes in the genome. Here, we used a heterologous expression system to characterize FtsZ and Cdv proteins fromN. maritimusby investigating the ability of these proteins to form polymers. We show that one of the Cdv proteins inN. maritimus, the CdvB (Nmar_0816), is capable of forming stable polymers when expressed in fission yeast. TheN. maritimusCdvB is also capable of assembling into filaments in mammalian cells. However,N. maritimusFtsZ does not assemble into polymers in our system. The ability of CdvB, but not FtsZ, to polymerize is consistent with a recent finding showing that several Cdv proteins, but not FtsZ, localize to the mid-cell site in the dividingN. maritimus. Thus, we propose that it is Cdv proteins, rather than FtsZ, that function as the cell division apparatus inN. maritimus.

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