Summary The key technical bottleneck for exploiting plant hairy root cultures as a robust bioproduction platform for therapeutic proteins has been low protein productivity, particularly low secreted protein yields. To address this, we engineered novel hydroxyproline (Hyp)‐ O ‐glycosylated peptides (Hyp GP s) into tobacco hairy roots to boost the extracellular secretion of fused proteins and to elucidate Hyp‐ O ‐glycosylation process of plant cell wall Hyp‐rich glycoproteins. Hyp GP s representing two major types of cell wall glycoproteins were examined: an extensin module consisting of 18 tandem repeats of ‘Ser‐Hyp‐Hyp‐Hyp‐Hyp’ motif or ( SP 4) 18 and an arabinogalactan protein module consisting of 32 tandem repeats of ‘Ser‐Hyp’ motif or ( SP ) 32 . Each module was expressed in tobacco hairy roots as a fusion to the enhanced green fluorescence protein ( EGFP ). Hairy root cultures engineered with a Hyp GP module secreted up to 56‐fold greater levels of EGFP , compared with an EGFP control lacking any Hyp GP module, supporting the function of Hyp GP modules as a molecular carrier in promoting efficient transport of fused proteins into the culture media. The engineered ( SP 4) 18 and ( SP ) 32 modules underwent Hyp‐ O ‐glycosylation with arabino‐oligosaccharides and arabinogalactan polysaccharides, respectively, which were essential in facilitating secretion of the fused EGFP protein. Distinct non‐Hyp‐ O ‐glycosylated ( SP 4) 18 ‐ EGFP and ( SP ) 32 ‐ EGFP intermediates were consistently accumulated within the root tissues, indicating a rate‐limiting trafficking and/or glycosylation of the engineered Hyp GP modules. An updated model depicting the intracellular trafficking, Hyp‐ O ‐glycosylation and extracellular secretion of extensin‐styled ( SP 4) 18 module and AGP ‐styled ( SP ) 32 module is proposed.

In vitro cultured plant cells, in particular the tobacco BY-2 cell, have demonstrated their potential to provide a promising bioproduction platform for therapeutic proteins by integrating the merits of whole-plant cultivation systems with those of microbial and mammalian cell cultures. Over the past three decades, substantial progress has been made in improving the plant cell culture system, resulting in a few commercial success cases, such as taliglucerase alfa (Elelyso ® ), the first FDA-approved recombinant pharmaceutical protein derived from plant cells. However, compared to the major expression hosts (bacteria, yeast, and mammalian cells), plant cells are still largely underutilized, mainly due to low productivity and non-human glycosylation. Modern molecular biology tools, in particular RNAi and the latest genome editing technology CRISPR/Cas9, have been used to modulate the genome of plant cells to create new cell lines that exhibit desired “traits” for producing therapeutic proteins. This review highlights the recent advances in cellular engineering of plant cells towards improved recombinant protein production, including creating cell lines with deficient protease levels or humanized glycosylation, and considers potential development in the future.

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Cyclase-associated protein 1 (CAP1) is a conserved actin-regulating protein that enhances actin filament dynamics and also regulates adhesion in mammalian cells. We previously found that phosphorylation at the Ser307/Ser309 tandem site controls its association with cofilin and actin and is important for CAP1 to regulate the actin cytoskeleton. Here, we report that transient Ser307/Ser309 phosphorylation is required for CAP1 function in both actin filament disassembly and cell adhesion. Both the phosphomimetic and the nonphosphorylatable CAP1 mutant, which resist transition between phosphorylated and dephosphorylated forms, had defects in rescuing the reduced rate of actin filament disassembly in the CAP1 knockdown HeLa cells. The phosphorylation mutants also had defects in alleviating the elevated focal adhesion kinase (FAK) activity and the enhanced focal adhesions in the knockdown cells. In dissecting further phosphoregulatory cell signals for CAP1, we found that cyclin-dependent kinase 5 (CDK5) phosphorylates both Ser307 and Ser309 residues, whereas cAMP signaling induces dephosphorylation at the tandem site, through its effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac). No evidence supports an involvement of activated protein phosphatase in executing the dephosphorylation downstream from cAMP, whereas preventing CAP1 from accessing its kinase CDK5 appears to underlie CAP1 dephosphorylation induced by cAMP. Therefore, this study provides direct cellular evidence that transient phosphorylation is required for CAP1 functions in both actin filament turnover and adhesion, and the novel mechanistic insights substantially extend our knowledge of the cell signals that function in concert to regulate CAP1 by facilitating its transient phosphorylation.

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had a profound impact on the world’s health and economy. Although the end of the pandemic may come in 2023, it is generally believed that the virus will not be completely eradicated. Most likely, the disease will become an endemicity. The rapid development of vaccines of different types (mRNA, subunit protein, inactivated virus, etc.) and some other antiviral drugs (Remdesivir, Olumiant, Paxlovid, etc.) has provided effectiveness in reducing COVID-19’s impact worldwide. However, the circulating SARS-CoV-2 virus has been constantly mutating with the emergence of multiple variants, which makes control of COVID-19 difficult. There is still a pressing need for developing more effective antiviral drugs to fight against the disease. Plants have provided a promising production platform for both bioactive chemical compounds (small molecules) and recombinant therapeutics (big molecules). Plants naturally produce a diverse range of bioactive compounds as secondary metabolites, such as alkaloids, terpenoids/terpenes and polyphenols, which are a rich source of countless antiviral compounds. Plants can also be genetically engineered to produce valuable recombinant therapeutics. This molecular farming in plants has an unprecedented opportunity for developing vaccines, antibodies, and other biologics for pandemic diseases because of its potential advantages, such as low cost, safety, and high production volume. This review summarizes the latest advancements in plant-derived drugs used to combat COVID-19 and discusses the prospects and challenges of the plant-based production platform for antiviral agents.

CAP1 ( Cyclase Associated Protein 1) is a conserved actin‐regulating protein that enhances actin filament dynamics and also regulates adhesion in mammalian cells. We previously found that phosphorylation at the Ser307/Ser309 tandem site controls its association with cofilin and actin, and is important for CAP1 to regulate the actin cytoskeleton. Here, we report that transient Ser307/Ser309 phosphorylation is required for both CAP1 functions in actin filament disassembly and cell adhesions. Both the phospho‐mimetic and non‐phosphorylatable CAP1 mutants, which resist transition between phosphorylated and dephosphorylated forms, had defects in rescuing the reduced rate of actin filament disassemblyin the CAP1‐knockdown HeLa cells. The phosphor mutants also had defects in alleviating the elevated FAK ( Focal Adhesion Kinase) activity and the enhanced focal adhesions in the knockdown cells. In dissecting further phosphor‐regulatory cell signals for CAP1, we found that CDK5 ( Cyclin‐ Dependent Kinase 5) phosphorylates both Ser307 and Ser309 residues, whereas cAMP signaling induces dephosphorylation at the tandem site, through its effectors PKA ( Protein Kinase A) and Epac ( Exchange proteins directly activated by cAMP). No evidence supports involvement of activated protein phosphatase in executing the dephosphorylation downstream of cAMP, whereas preventing CAP1 access to its kinase CDK5 appears to underlie CAP1 dephosphorylation induced by cAMP. Therefore, this study provides direct cellular evidence that transient phosphorylation is required for both CAP1 functions in actin filament turnover and adhesion, and the novel mechanistic insights substantially extend our knowledge on the cell signals that function in concert to regulate CAP1 by facilitating its transient phosphorylation. Support or Funding Information This project was supported by an Institutional Development Award(IDeA) from the National Institute of General Medical Sciencesat the National Institutes of Health [Grant number: P20GM103429].