Oxidative Folding of Proteins: The “Smoking Gun” of Glutathione

Glutathione has long been suspected to be the primary low molecular weight compound present in all cells promoting the oxidative protein folding, but twenty years ago it was found “not guilty”. Now, new surprising evidence repeats its request to be the “smoking gun” which reopens the criminal trial revealing the crucial involvement of this tripeptide.

The yeast eIF2 kinase Gcn2 facilitates H 2 O 2 -mediated feedback inhibition of both protein synthesis and ER oxidative folding during recombinant protein production

Recombinant protein production is a known source of oxidative stress. Knowledge of which ROS are involved or the specific growth phase in which stress occurs however remains lacking. Using modern, hypersensitive genetic H 2 O 2 -specific probes, micro-cultivation and continuous measurements in batch culture, we observed H 2 O 2 accumulation during and following the diauxic shift in engineered Saccharomyces cerevisiae , correlating with peak α-amylase production. In agreement with previous studies supporting a role of the translation initiation factor kinase Gcn2 in the response to H 2 O 2 , we find Gcn2-dependent phosphorylation of eIF2α to increase alongside translational attenuation in strains engineered to produce large amounts of α-amylase. Gcn2 removal significantly improved α-amylase production in two previously optimized high-producing strains, but not in the wild-type. Gcn2-deficiency furthermore reduced intracellular H 2 O 2 levels and the Hac1 splicing ratio whilst expression of antioxidants and the ER disulfide isomerase PDI1 increased. These results suggest protein synthesis and ER oxidative folding to be coupled and subject to feedback inhibition by H 2 O 2 . Importance Recombinant protein production is a multi-billion dollar industry. Optimizing the productivity of host cells is, therefore, of large interest. In several hosts oxidants are produced as an unwanted side product of recombinant protein production. The buildup of oxidants can result in intracellular stress responses which could compromise the productivity of the host cell. Here we document a novel protein synthesis inhibitory mechanism that is activated by the buildup of a specific oxidant (H 2 O 2 ) in the cytosol of yeast cells upon the production of recombinant proteins. At the center of this inhibitory mechanism lies the protein kinase Gcn2. By removing Gcn2 we observed a doubling of recombinant protein productivity in addition to reduced H 2 O 2 levels in the cytosol. By this study we want to raise awareness of this inhibitory mechanism in eukaryotic cells to further improve protein production and contribute to the development of novel protein-based therapeutic strategies.

A VHH-Fc Fusion Targeted to the Chloroplast Thylakoid Lumen Assembles and Neutralizes Enterohemorrhagic E. coli O157:H7

Chimeric fusion proteins comprising a single domain antibody (VHH) fused to a crystallizable fragment (Fc) of an immunoglobulin are modular glycoproteins that are becoming increasingly in demand because of their value as diagnostics, research reagents and passive immunization therapeutics. Because ER-associated degradation and misfolding may potentially be limiting factors in the oxidative folding of VHH-Fc fusion proteins in the ER, we sought to explore oxidative folding in an alternative sub-compartment, the chloroplast thylakoid lumen, and determine its viability in a molecular farming context. We developed a set of in-house expression vectors for transient transformation of Nicotiana benthamiana leaves that target a VHH-Fc to the thylakoid lumen via either secretory (Sec) or twin-arginine translocation (Tat) import pathways. Compared to stromal [6.63 ± 3.41 mg/kg fresh weight (FW)], cytoplasmic (undetectable) and Tat-import pathways (5.43 ± 2.41 mg/kg FW), the Sec-targeted VHH-Fc showed superior accumulation (30.56 ± 5.19 mg/kg FW), but was less than that of the ER (51.16 ± 9.11 mg/kg FW). Additionally, the introduction of a rationally designed de novo disulfide bond enhances in planta accumulation when introduced into the Sec-targeted Fc fusion protein from 50.24 ± 4.08 mg/kg FW to 110.90 ± 6.46 mg/kg FW. In vitro immunofluorescent labeling assays on VHH-Fc purified from Sec, Tat, and stromal pathways demonstrate that the antibody still retains VHH functionality in binding Escherichia coli O157:H7 and neutralizing its intimate adherence to human epithelial type 2 cells. These results overall provide a proof of concept that the oxidative folding environment of the thylakoid lumen may be a viable compartment for stably folding disulfide-containing recombinant VHH-Fc proteins.

Ca2+ Regulates ERp57-Calnexin Complex Formation

ERp57, a member of the protein disulfide isomerase family, is a ubiquitous disulfide catalyst that functions in the oxidative folding of various clients in the mammalian endoplasmic reticulum (ER). In concert with ER lectin-like chaperones calnexin and calreticulin (CNX/CRT), ERp57 functions in virtually all folding stages from co-translation to post-translation, and thus plays a critical role in maintaining protein homeostasis, with direct implication for pathology. Here, we present mechanisms by which Ca2+ regulates the formation of the ERp57-calnexin complex. Biochemical and isothermal titration calorimetry analyses revealed that ERp57 strongly interacts with CNX via a non-covalent bond in the absence of Ca2+. The ERp57-CNX complex not only promoted the oxidative folding of human leukocyte antigen heavy chains, but also inhibited client aggregation. These results suggest that this complex performs both enzymatic and chaperoning functions under abnormal physiological conditions, such as Ca2+ depletion, to effectively guide proper oxidative protein folding. The findings shed light on the molecular mechanisms underpinning crosstalk between the chaperone network and Ca2+.

The Mia40/CHCHD4 Oxidative Folding System: Redox Regulation and Signaling in the Mitochondrial Intermembrane Space

Mitochondria are critical for several cellular functions as they control metabolism, cell physiology, and cell death. The mitochondrial proteome consists of around 1500 proteins, the vast majority of which (about 99% of them) are encoded by nuclear genes, with only 13 polypeptides in human cells encoded by mitochondrial DNA. Therefore, it is critical for all the mitochondrial proteins that are nuclear-encoded to be targeted precisely and sorted specifically to their site of action inside mitochondria. These processes of targeting and sorting are catalysed by protein translocases that operate in each one of the mitochondrial sub-compartments. The main protein import pathway for the intermembrane space (IMS) recognises proteins that are cysteine-rich, and it is the only import pathway that chemically modifies the imported precursors by introducing disulphide bonds to them. In this manner, the precursors are trapped in the IMS in a folded state. The key component of this pathway is Mia40 (called CHCHD4 in human cells), which itself contains cysteine motifs and is subject to redox regulation. In this review, we detail the basic components of the MIA pathway and the disulphide relay mechanism that underpins the electron transfer reaction along the oxidative folding mechanism. Then, we discuss the key protein modulators of this pathway and how they are interlinked to the small redox-active molecules that critically affect the redox state in the IMS. We present also evidence that the mitochondrial redox processes that are linked to iron–sulfur clusters biogenesis and calcium homeostasis coalesce in the IMS at the MIA machinery. The fact that the MIA machinery and several of its interactors and substrates are linked to a variety of common human diseases connected to mitochondrial dysfunction highlight the potential of redox processes in the IMS as a promising new target for developing new treatments for some of the most complex and devastating human diseases.

The yeast eIF2 kinase Gcn2 facilitates H2O2-mediated feedback inhibition of both protein synthesis and ER oxidative folding during recombinant protein production

Recombinant protein production is a known source of oxidative stress. Knowledge of which ROS are involved or the specific growth phase in which stress occurs however remains lacking. Using modern, hypersensitive genetic H2O2-specific probes, micro-cultivation and continuous measurements in batch culture, we observed H2O2 accumulation during and following the diauxic shift in engineered Saccharomyces cerevisiae, correlating with peak α-amylase production. In agreement with previous studies supporting a role of the translation initiation factor kinase Gcn2 in the response to H2O2, we find Gcn2-dependent phosphorylation of eIF2α to increase alongside translational attenuation in strains engineered to produce large amounts of α-amylase. Gcn2 removal significantly improved α-amylase production in two previously optimized high-producing strains, but not in the wild-type. Gcn2-deficiency furthermore reduced intracellular H2O2 levels and the unfolded protein response whilst expression of antioxidants and the ER disulfide isomerase PDI1 increased. These results suggest protein synthesis and ER oxidative folding to be coupled and subject to feedback inhibition by H2O2.ImportanceReactive oxygen species (ROS) accumulate during recombinant protein production both in yeast and Chinese hamster ovary cells, two of the most popular organisms used in the multi-million dollar protein production industry. Here we document increased H2O2 in the cytosol of yeast cells producing α-amylase. Since H2O2 predominantly targets the protein synthesis machinery and activates the translation initiation factor kinase Gcn2, we removed Gcn2, resulting in increased recombinant α-amylase production in two different previously engineered high-producing protein production strains. Removal of this negative feed-back loop thus represents a complementary strategy for improving recombinant protein production efforts currently used in yeast. Gcn2-deficiency also increased the expression of antioxidant genes and the ER-foldase PDI1, suggesting that protein synthesis and ER oxidative folding are linked and feed-back regulated via H2O2. Identification of additional components in this complex regulation may further improve protein production and contribute to the development of novel protein-based therapeutic strategies.