Overexpression of human ACE2 protein in mouse fibroblasts stably transfected with the intact ACE2 gene.

Infection by SARS-CoV-2 is dependent on binding of the viral spike protein to angiotensin converting enzyme 2 (ACE2), a membrane glycoprotein expressed on epithelial cells in the human upper respiratory tract. Recombinant ACE2 protein has potential application for anti-viral therapy. Here we co-transfected mouse fibroblasts (A9 cells) with a cloned fragment of human genomic DNA containing the intact ACE2 gene and an unlinked neomycin phosphotransferase gene, and then selected stable neomycin-resistant transfectants. Transfectant clones expressed ACE2 protein at levels that were generally proportional to the number of ACE2 gene copies integrated in the cell genome, ranging up to approximately 50 times the level of ACE2 present of Vero-E6 cells. Cells overexpressing ACE2 were hypersensitive to infection by spike-pseudotyped vesicular stomatitis virus (VSV-S), and adsorption of VSV-S to these cells occurred at an accelerated rate compared to Vero-E6 cells. The transfectant cell clones described here therefore have favorable attributes as feedstocks for large-scale production of recombinant human ACE2 protein.

A polypeptide model for toxic aberrant proteins induced by aminoglycoside antibiotics.

Aminoglycoside antibiotics interfere with the selection of cognate tRNAs during translation, resulting in the synthesis of aberrant proteins that are the ultimate cause of cell death. However, the toxic potential of aberrant proteins and how they avoid degradation by the cell's protein quality control (QC) machinery are not understood. Here we report that levels of the heat shock (HS) transcription factor σ32 increased sharply following exposure of Escherichia coli to the aminoglycoside kanamycin (Kan), suggesting that at least some of the aberrant proteins synthesized in these cells were recognized as substrates by DnaK, a molecular chaperone that regulates the HS response, the major protein QC pathway in bacteria. To further investigate aberrant protein toxic potential and interaction with cell QC factors, we studied an acutely toxic 48-residue polypeptide (ARF48) that is encoded by an alternate reading frame in a plant cDNA. As occurred in cells exposed to Kan, σ32 levels were strongly elevated following ARF48 expression, suggesting that ARF48 was recognized as a substrate by DnaK. Paradoxically, an internal 10-residue region that was tightly bound by DnaK in vitro also was required for the ARF48 toxic effect. Despite the increased levels of σ32, levels of several HS proteins were unchanged following ARF48 expression, suggesting that the HS response had been aborted. Nucleoids were condensed and cell permeability increased rapidly following ARF48 expression, together suggesting that ARF48 disrupts DNA-membrane interactions that could be required for efficient gene expression. Our results are consistent with earlier studies showing that aberrant proteins induced by aminoglycoside antibiotics disrupt cell membrane integrity. Insights into the mechanism for this effect could be gained by further study of the ARF48 model system.

Regulation of a viral proteinase by a peptide and DNA in one-dimensional space: I. binding to DNA AND to hexon of the precursor to protein VI, pVI, of human adenovirus.

The precursor to adenovirus protein VI, pVI, is a multifunctional protein with different roles early and late in virus infection. Here, we focus on two roles late in infection, binding of pVI to DNA and to the major capsid protein hexon. pVI bound to DNA as a monomer independent of DNA sequence with an apparent equilibrium dissociation constant, K(d)((app)), of 46 nm. Bound to double-stranded DNA, one molecule of pVI occluded 8 bp. Upon the binding of pVI to DNA, three sodium ions were displaced from the DNA. A ΔG(0)(0) of -4.54 kcal/mol for the nonelectrostatic free energy of binding indicated that a substantial component of the binding free energy resulted from nonspecific interactions between pVI and DNA. The proteolytically processed, mature form of pVI, protein VI, also bound to DNA; its K(d)((app)) was much higher, 307 nm. The binding assays were performed in 1 mm MgCl(2) because in the absence of magnesium, the binding to pVI or protein VI to DNA was too tight to determine a K(d)((app)). Three molecules of pVI bound to one molecule of the hexon trimer with an equilibrium dissociation constant K(d)((app)) of 1.1 nm.

Toxic misfolding of Arabidopsis cellulases in the secretory pathway of Pichia pastoris.

Plants produce a large number of cellulases that are either secreted or anchored in the plasma membrane where they likely function in various aspects of cellulose synthesis, modification and degradation during plant growth and development. Very few of these enzymes have been characterized in any detail, however. Here we attempted to produce two Arabidopsis modular cellulases, which contain a catalytic domain belonging to glycoside hydrolase family 9 (GH9) and a carbohydrate binding module (CBM), in the yeast Pichia pastoris. Neither of the intact modular enzymes was detectably produced, although the independently expressed GH9 catalytic domain of one enzyme was secreted when the protein was expressed at low temperature. Expression of intact and truncated cellulases at the standard temperature caused extensive cell lysis, with release of high concentrations of endogenous proteins into the culture medium. Cell lysis appeared to result from misfolding of cellulase proteins within the Pichia secretory pathway. The toxicity of these misfolded cellulases potentially could be exploited to derive host strains with enhanced capability to fold recombinant secretory proteins.

Export is the default pathway for soluble unfolded polypeptides that accumulate during expression in Escherichia coli.

Several E. coli endogenous, cytoplasmic proteins that are known clients of the chaperonin GroEL were overexpressed to examine the fate of accumulated unfolded polypeptides. Substantial fractions of about half of the proteins formed insoluble aggregates, consistent with the hypothesis that these proteins were produced at rates or in amounts that exceeded the protein-folding capacity of GroEL. In addition, large fractions of three overexpressed GroEL client proteins were localized in an extra-cytoplasmic, osmotically-sensitive compartment, suggesting they had initially accumulated in the cytoplasm as soluble unfolded polypeptides and thus were able to access a protein export pathway. Consistent with this model, an intrinsically unfoldable, hydrophilic, non-secretory polypeptide was quantitatively exported from the E. coli cytoplasm into an osmotically-sensitive compartment. Our results support the conclusion that a soluble, unfolded conformation alone may be sufficient to direct non-secretory polypeptides into a protein export pathway for signal peptide-independent translocation across the inner membrane, and that export rather than degradation by cytoplasmic proteases is the preferred fate for newly-synthesized, soluble, unfolded polypeptides that accumulate in the cytoplasm. The stable folded conformation of exported GroEL client proteins further suggests that the requirement for GroEL may be conditional on protein folding in the molecularly-crowded environment of the cytoplasm.

Thiamine diphosphate binds to intermediates in the assembly of adenovirus fiber knob trimers in Escherichia coli.

Assembly of the adenovirus (Ad) homotrimeric fiber protein is nucleated by its C-terminal knob domain, which itself can trimerize when expressed as a recombinant protein fragment. The non-interlocked, globular structure of subunits in the knob trimer implies that trimers assemble from prefolded monomers through a dimer intermediate, but these intermediates have not been observed and the mechanism of assembly therefore remains uncharacterized. Here we report that expression of the Ad serotype 2 (Ad2) knob was toxic for thi- strains of Escherichia coli, which are defective in de novo synthesis of thiamine (vitamin B1). Ad2 knob trimers isolated from a thi+ strain copurified through multiple chromatography steps with a small molecule of mass equivalent to that of thiamine diphosphate (ThDP). Mutant analysis did not implicate any specific site for ThDP binding. Our results suggest that ThDP may associate with assembly intermediates and become trapped in assembled trimers, possibly within one of several large cavities that are partially solvent-accessible or buried completely within the trimer interior.

Functionalized carbon nanotubes for detecting viral proteins.

We investigated the biocompatibility, specificity, and activity of a ligand-receptor-protein system covalently bound to oxidized single-walled carbon nanotubes (SWNTs) as a model proof-of-concept for employing such SWNTs as biosensors. SWNTs were functionalized under ambient conditions with either the Knob protein domain from adenovirus serotype 12 (Ad 12 Knob) or its human cellular receptor, the CAR protein, via diimide-activated amidation. We confirmed the biological activity of Knob protein immobilized on the nanotube surfaces by using its labeled conjugate antibody and evaluated the activity and specificity of bound CAR on SWNTs, first, in the presence of fluorescently labeled Knob, which interacts specifically with CAR, and second, with a negative control protein, YieF, which is not recognized by biologically active CAR proteins. In addition, current-gate voltage (I-V(g)) measurements on a dozen nanotube devices explored the effect of protein binding on the intrinsic electronic properties of the SWNTs, and also demonstrated the devices' high sensitivity in detecting protein activity. All data showed that both Knob and CAR immobilized on SWNT surfaces fully retained their biological activities, suggesting that SWNT-CAR complexes can serve as biosensors for detecting environmental adenoviruses.

Protein aggregation during overexpression limited by peptide extensions with large net negative charge.

Folding of the human coxsackie and adenovirus receptor immunoglobulin (Ig) variable-type domain (CAR D1) during overexpression in the Escherichia coli cytoplasm was shown previously to be partially rescued by fusion to a 22-residue C-terminal peptide. Here, peptide sequence features required for solubilization and folding of CAR D1 and similar Ig variable-type domains from two other human membrane proteins were investigated. Peptide extensions with net negative charge > -6 fully solubilized CAR D1, and approximately half of the peptide-solubilized protein was correctly folded. The Ig variable-type domains from human A33 antigen and myelin P-zero proteins were only partially solubilized by peptide extensions with net charge of -12, however, and only the solubilized P-zero domain appeared to fold correctly whereas the A33 domain formed soluble microaggregates of misfolded protein. Our results suggest a model where the large net charge of peptide extensions increases electrostatic repulsion between nascent polypeptides. The resulting decrease in aggregation rate can enable some polypeptides to fold spontaneously into their native protein conformations. Analysis of the solubility and folding status of sets of structurally homologous proteins, such as the Ig variable-type domains described here, during overexpression could provide insights into how amino acid and gene sequences influence the efficiency of spontaneous protein folding.

Biodistribution of radioiodinated adenovirus fiber protein knob domain after intravenous injection in mice.

The knob domains from the fiber proteins of adenovirus serotypes 2 and 12 were labeled with radioiodine and then injected into the bloodstreams of mice. Knob proteins with functional binding sites for the coxsackie and adenovirus receptor (CAR) were cleared rapidly from the circulation, with radioactivity appearing predominantly in the stomach, while knob mutants unable to bind to CAR remained in the blood circulation for a prolonged period. The clearance of radiolabeled wild-type knob from the blood was slowed by coinjecting an excess of unlabeled wild-type knob protein. An earlier study showed that (99m)Tc-labeled knob protein with intact CAR-binding activity also cleared rapidly from the blood circulation of mice, with radioactivity accumulating predominantly in the liver (K. R. Zinn et al., Gene Ther. 5:798-808, 1998). Together these results suggest that rapid clearance of knob protein from the blood results from specific binding to CAR in the liver and that the bound knob then enters a degradative pathway. The elevated levels of radioiodine in the stomach observed in our experiments are consistent with deiodination of labeled knob by dehalogenases in hepatocyte microsomes and uptake of the resultant free radioiodine by Na/I symporters in the gastric mucosa. Although CAR has been shown to localize in tight junctions of polarized epithelial cells, where it functions in intercellular adhesion, the results of our study suggest that a subset of CAR molecules in the liver is highly accessible to ligands in the blood and able to rapidly deliver bound ligand to an intracellular degradative compartment.

Structural basis for variation in adenovirus affinity for the cellular coxsackievirus and adenovirus receptor.

The majority of adenovirus serotypes can bind to the coxsackievirus and adenovirus receptor (CAR) on human cells despite only limited conservation of the amino acid residues that comprise the receptor-binding sites of these viruses. Using a fluorescence anisotropy-based assay, we determined that the recombinant knob domain of the fiber protein from adenovirus serotype (Ad) 2 binds the soluble, N-terminal domain (domain 1 (D1)) of CAR with 8-fold greater affinity than does the recombinant knob domain from Ad12. Homology modeling predicted that the increased affinity of Ad2 knob for CAR D1 could result from additional contacts within the binding interface contributed by two residues, Ser408 and Tyr477, which are not conserved in the Ad12 knob. Consistent with this structural model, substitution of serine and tyrosine for the corresponding residues in the Ad12 knob (P417S and S489Y) increased the binding affinity by 4- and 8-fold, respectively, whereas the double mutation increased binding affinity 10-fold. X-ray structure analysis of Ad12 knob mutants P417S and S489Y indicated that both substituted residues potentially could form additional hydrogen bonds across the knob-CAR interface. Structural changes resulting from these mutations were highly localized, implying that the high tolerance for surface variation conferred by the stable knob scaffold can minimize the impact of antigenic drift on binding specificity and affinity during evolution of virus serotypes. Our results suggest that the interaction of knob domains from different adenovirus serotypes with CAR D1 can be accurately modeled using the Ad12 knob-CAR D1 crystal structure as a template.

Adenovirus fiber disrupts CAR-mediated intercellular adhesion allowing virus escape.

Adenovirus binds its receptor (CAR), enters cells, and replicates. It must then escape to the environment to infect a new host. We found that following infection, human airway epithelia first released adenovirus to the basolateral surface. Virus then traveled between epithelial cells to emerge on the apical surface. Adenovirus fiber protein, which is produced during viral replication, facilitated apical escape. Fiber binds CAR, which sits on the basolateral membrane where it maintains tight junction integrity. When fiber bound CAR, it disrupted junctional integrity, allowing virus to filter between the cells and emerge apically. Thus, adenovirus exploits its receptor for two important but distinct steps in its life cycle: entry into host cells and escape across epithelial barriers to the environment.