Inactivation of SARS-CoV-2 and HCoV-229E in vitro by ColdZyme® a medical device mouth spray against the common cold.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic calls for effective and safe treatments. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing COVID-19 actively replicates in the throat, unlike SARS-CoV, and shows high pharyngeal viral shedding even in patients with mild symptoms of the disease. HCoV-229E is one of four coronaviruses causing the common cold. In this study, the efficacy of ColdZyme® (CZ-MD), a medical device mouth spray, was tested against SARS-CoV-2 and HCoV-229E in vitro. The CZ-MD provides a protective glycerol barrier containing cod trypsin as an ancillary component. Combined, these ingredients can inactivate common cold viruses in the throat and mouth. The CZ-MD is believed to act on the viral surface proteins that would perturb their entry pathway into cells. The efficacy and safety of the CZ-MD have been demonstrated in clinical trials on the common cold. METHOD OF STUDY: The ability of the CZ-MD to inactivate SARS-CoV-2 and HCoV-229E was tested using an in vitro virucidal suspension test (ASTM E1052). RESULTS: CZ-MD inactivated SARS-CoV-2 by 98.3% and HCoV-229E by 99.9%. CONCLUSION: CZ-MD mouth spray can inactivate the respiratory coronaviruses SARS-CoV-2 and HCoV-229E in vitro. Although the in vitro results presented cannot be directly translated into clinical efficacy, the study indicates that CZ-MD might offer a protective barrier against SARS-CoV-2 and a decreased risk of COVID-19 transmission.

Biochemical characterization of a native group III trypsin ZT from Atlantic cod (Gadus morhua).

Atlantic cod trypsin ZT is biochemically characterized for the first time in this report in comparison to a group I trypsin (cod trypsin I). To our knowledge, trypsin ZT is the first thoroughly characterized group III trypsin. A more detailed understanding of trypsin ZT biochemistry may give insight into its physiological role as well as its potential use within the biotechnology sector. Stability is an important factor when it comes to practical applications of enzymes. Compared to trypsin I, trypsin ZT shows differences in pH and heat stability, sensitivity to inhibitors and sub-site substrate specificity as shown by multiplex substrate profiling analysis. Based on the analysis, trypsin ZT cleaved at arginine and lysine as other trypsins. Furthermore, trypsin ZT is better than trypsin I in cleaving peptides containing several consecutive positively charged residues. Lysine- and arginine-rich amino acid sequences are frequently found in human viral proteins. Thus, trypsin ZT may be effective in inactivating human and fish viruses implying a possible role for the enzyme in the natural defence of Atlantic cod. The results from this study can lead to multiple practical applications of trypsin ZT.

Elucidation of different cold-adapted Atlantic cod (Gadus morhua) trypsin X isoenzymes.

Trypsins from Atlantic cod (Gadus morhua), consisting of several isoenzymes, are highly active cold-adapted serine proteases. These trypsins are isolated for biomedical use in an eco-friendly manner from underutilized seafood by-products. Our group has explored the biochemical properties of trypsins and their high potential in biomedicine. For broader utilization of cod trypsins, further characterization of biochemical properties of the individual cod trypsin isoenzymes is of importance. For that purpose, a benzamidine purified trypsin isolate from Atlantic cod was analyzed. Anion exchange chromatography revealed eight peaks containing proteins around 24kDa with tryptic activity. Based on mass spectrometric analysis, one isoenzyme gave the best match to cod trypsin I and six isoenzymes gave the best match to cod trypsin X. Amino terminal sequencing of two of these six trypsin isoenzymes showed identity to cod trypsin X. Three sequence variants of trypsin X were identified by cDNA analysis demonstrating that various forms of this enzyme exist. One trypsin X isoenzyme was selected for further characterization based on abundance and stability. Stepwise increase in catalytic efficiency (kcat/Km) of this trypsin X isoenzyme was obtained with substrates containing one to three amino acid residues. The study demonstrates that the catalytic efficiency of this trypsin X isoenzyme is comparable to that of cod trypsin I, the most abundant and highly active isoenzyme in the benzamidine cod trypsin isolate. Differences in pH stability and sensitivity to inhibitors of the trypsin X isoenzyme compared to cod trypsin I were detected that may be important for practical use.

Potential use of Atlantic cod trypsin in biomedicine.

Surface proteins of viruses and bacteria used for cell attachment and invasion are candidates for degradation by proteases. Trypsin from Atlantic cod (Gadus morhua) was previously demonstrated to have efficacy against influenza viruses in vitro and on skin. In this paper, cod trypsin is shown to be 3-12 times more effective in degrading large native proteins than its mesophilic analogue, bovine trypsin. This is in agreement with previous findings where cod trypsin was found to be the most active among twelve different proteases in cleaving various cytokines and pathological proteins. Furthermore, our results show that cod trypsin has high efficacy against herpes simplex virus type 1 (HSV-1) and the respiratory syncytial virus (RSV) in vitro. The results on the antipathogenic properties of cod trypsin are important because rhinovirus, RSV, and influenza are the most predominant pathogenic viruses in upper respiratory tract infections. Results from a clinical study presented in this paper show that a specific formulation containing cod trypsin was preferred for wound healing over other methods used in the study. Apparently, the high digestive ability of the cold-adapted cod trypsin towards large native proteins plays a role in its efficacy against pathogens and its positive effects on wounds.

Characterization of cold-adapted Atlantic cod (Gadus morhua) trypsin I--kinetic parameters, autolysis and thermal stability.

Atlantic cod trypsin I is a highly active cold-adapted protease. This study aimed at further characterization of this enzyme with respect to kinetic parameters, sites of autolysis and stability. For that purpose, trypsin I was purified by anion exchange chromatography. Its purity and identity was verified by SDS-PAGE analysis and mass spectrometry. Concomitantly, another cod trypsin isozyme, trypsin X, previously only described from its cDNA sequence was detected in a separate peak from the ion exchange chromatogram. There was a stepwise increase in the catalytic efficiency (k(cat)/K(m)) of cod trypsin I obtained with substrates containing one to three amino acid residues. As expected, the activity of trypsin I was maintained for longer periods of time at 15 degrees C than at higher temperatures. The residues of the trypsin I molecule most sensitive to autolysis were identified using Edman degradation. Eleven autolytic cleavage sites were detected within the trypsin I molecule. Unfolding experiments demonstrated that autolysis is a contributing factor in the stability of trypsin I. In addition, the data shows that cod trypsin I is less stable towards thermal unfolding than its mesophilic bovine analogue.

Protein phosphatase 6 regulatory subunits composed of ankyrin repeat domains.

Protein phosphatase 6 (PP6) is an essential Ser/Thr phosphatase conserved among eukaryotes. The Saccharomyces cerevisiae homologue of PP6 called Sit4 depends on association with SAPS domain subunits. This study used a human SAPS domain subunit FLAG-PP6R1 to identify endogenous interacting proteins. Mass spectrometry identified coprecipitating proteins as PP6 catalytic subunit and three ankyrin repeat proteins (Ankrd28, Ankrd44, and Ankrd52). These proteins have extensive sequence identity to one another but segregate into separate branches on a phylogenetic tree for vertebrate species, suggesting individual biological functions. Tagged Ankrd28 coprecipitated with PP6, not with PP2A or PP4, and with SAPS domain subunits PP6R1 and PP6R3. Tagged PP6 coprecipitated endogenous SAPS domain subunits and Ankrd28. The C-terminal region of PP6R1 was sufficient to coprecipitate Ankrd28, but not PP6, demonstrating that PP6R1 acts as a scaffold with separate regions for binding to PP6 and to Ankrd28. Endogenous PP6 holoenzymes with PP6R1 and PP6R3 subunits were resolved by DEAE chromatography and eluted together with Ankrd28 at Mr > 440 kDa from Superose 12. Knockdown of PP6R1 or Ankrd28, but not PP6R3, produced equivalent enhancement of IkappaBepsilon degradation in response to TNFalpha. The results suggest that PP6 functions as a heterotrimer, composed of the PP6 catalytic subunit bound to a SAPS domain scaffold subunit that associates with Ankrd28. We propose that the SAPS and ankyrin repeat regulatory subunits determine the function and specificity of PP6.

Protein phosphatase PP6 N terminal domain restricts G1 to S phase progression in human cancer cells.

Yeast SIT4 is an essential gene encoding a protein Ser/Thr phosphatase conserved throughout eukaryotic evolution, known as PPV in Drosophila and PP6 in vertebrates. Sit4 promotes transcription of G1 cyclins and a sit4(ts) strain exhibits a G1 arrest at the restrictive temperature. The yeast sit4(ts) was rescued by expression of PPV or a chimeric phosphatase containing the first fifty-three residues of PPV fused to Drosophila PP1. The results suggested that the N terminus of the Sit4/PPV protein exerts a specific function in the yeast cell cycle. Here we tested whether the N terminus of human PP6 exerts specific effects on G1-S progression in human cells. The N terminus of PP6 or PP2A was fused to GFP and the proteins transiently expressed in prostate cancer PC-3 cells. Expression of the PP6 fusion protein was restricted to lower levels than either the PP2A fusion protein or GFP. However, the PP6 fusion protein blocked entry into S phase and increased by >20% the proportion of cells in G1 phase. Expression of the PP6 fusion protein did not significantly change the levels of transcripts for cyclins or ca. eighty other cell cycle genes, but did suppress the levels of cyclin D1 protein in cells in G1 phase and reduce the phosphorylation of RB1 at Ser807/811. Thus, our results provide evidence that PP6 regulates cell cycle progression in human cells at least in part through control of cyclin D1 and the function of PP6 is distinct from its homolog Sit4 in yeast.

Protein phosphatase 6 down-regulates TAK1 kinase activation in the IL-1 signaling pathway.

TAK1 (transforming growth factor beta-activated kinase 1) is a serine/threonine kinase that is a mitogen-activated protein kinase kinase kinase and an essential intracellular signaling component in inflammatory signaling pathways. Upon stimulation of cells with inflammatory cytokines, TAK1 binds proteins that stimulate autophosphorylation within its activation loop and is thereby catalytically activated. This activation is transient; it peaks within a couple of minutes and is subsequently down-regulated rapidly to basal levels. The mechanism of down-regulation of TAK1 has not yet been elucidated. In this study, we found that toxin inhibition of type 2A protein phosphatases greatly enhances interleukin 1 (IL-1)-dependent phosphorylation of Thr-187 in the TAK1 activation loop as well as the catalytic activity of TAK1. From proteomic analysis of TAK1-binding proteins, we identified protein phosphatase 6 (PP6), a type-2A phosphatase, and demonstrated that PP6 associated with and inactivated TAK1 by dephosphorylation of Thr-187. Ectopic and endogenous PP6 co-precipitated with TAK1, and expression of PP6 reduced IL-1 activation of TAK1 but did not affect osmotic activation of MLK3, another MAPKKK. Reduction of PP6 expression by small interfering RNA enhances IL-1-induced phosphorylation of Thr-187 in TAK1. Enhancement occurred without change in levels of PP2A showing specificity for PP6. Our results demonstrate that PP6 specifically down-regulates TAK1 through dephosphorylation of Thr-187 in the activation loop, which is likely important for suppressing inflammatory responses via TAK1 signaling pathways.

Protein phosphatase 6 subunit with conserved Sit4-associated protein domain targets IkappaBepsilon.

Protein Ser/Thr phosphatases compose a PPP family that includes type-2 PP2A, PP4, and PP6, each with essential functions. The human PP6 gene rescues sit4(ts) mutants of Saccharomyces cerevisiae, and Sit4 phosphatase function depends on multiple Sit4-associated protein (SAP) subunits. We report here finding a SAPS sequence domain encoded in only a single gene each in Schizosaccharomyces pombe, Caenorhabditis elegans, and Drosophila but in three distinct open reading frames in Xenopus, Mus musculus, and Homo sapiens. The SAPS proteins are more divergent in sequence than PP6. Northern hybridization showed differential distribution of the human SAPS-related mRNA in multiple human tissues, named as PP6R1, PP6R2, and PP6R3. Antibodies were generated, distribution of endogenous PP6, PP6R1, PP6R2, and PP6R3 proteins was examined by immunoblotting, and the abundance of mRNA and protein in various tissues did not match. FLAG-tagged PP6R1 and PP6R2 expressed in HEK293 cells co-precipitated endogenous PP6, but not PP2A or PP4, showing specificity for recognition of phosphatases. The SAPS domain of PP6R1 alone was sufficient for association with PP6, and this predicts that conserved sequence motifs in the SAPS domain accounts for the specificity. FLAG-PP6R1 and FLAG-PP6R2 co-precipitated HA-IkappaBepsilon. Knockdown of PP6 or PP6R1 but not PP6R3 with siRNA significantly enhanced degradation of endogenous IkappaBepsilon in response to tumor necrosis factor-alpha. The results show SAPS domain subunits recruit substrates such as IkappaBepsilon as one way to determine specific functions for PP6.

Phosphorylation of the Pro-X-Thr-Pro site in phosphatase inhibitor-2 by cyclin-dependent protein kinase during M-phase of the cell cycle.

Protein phosphorylation serves as a primary mechanism for triggering events during mitosis and depends on coordinated regulation of kinases and phosphatases. Protein Ser-Thr phosphatase-1 (PP1) activity is essential for the metaphase to anaphase transition and the most ancient regulator of PP1 conserved from yeast to human is inhibitor-2 (I-2), an unstructured heat-stable protein. A unique sequence motif in I-2 from various species surrounds a phosphorylation site PXTP that can be phosphorylated in biochemical assays by GSK3, MAPK and CDK kinases. Here we used a phosphosite specific antibody to investigate the phosphorylation of I-2. We fractioned extracts from HeLa cells arrested with nocodazole and assayed for PXTP kinases using recombinant I-2. One major and two minor peaks of kinase activity were identified and the major peak contained both active MAPK and cdk1::cyclinB1, confirmed by immunoblotting. Cells released from a double thymidine block synchronously progressed through mitosis and immunoblotting revealed transient phosphorylation of endogenous I-2 in cells only during mitosis, and corresponding phosphorylation of histone H3 (Ser10) and PP1 (Thr320). Activation of cdk1::cyclinB1 was coincident with I-2 phosphorylation, but neither MAPK nor GSK3 were phosphorylated at this time, so we concluded that in living cells only cdk1::cyclinB1 phosphorylated the PXTP site in I-2. Immunofluorescent staining of cells with the PXTP phosphosite antibody revealed highly specific staining of mitotic cells prior to anaphase, at which point the staining disappeared. Thus, phosphorylation of I-2 is catalyzed by cdk1::cyclinB1 and staining with a specific antibody should prove useful as a selective marker of cells in the early stages of mitosis.

Phosphorylation of protein phosphatase type-1 inhibitory proteins by integrin-linked kinase and cyclic nucleotide-dependent protein kinases.

Protein phosphatases play key roles in cellular regulation and are subjected to control by protein inhibitors whose activity is in turn regulated by phosphorylation. Here we investigated the possible regulation of phosphorylation-dependent type-1 protein phosphatase (PP1) inhibitors, CPI-17, PHI-1, and KEPI, by various kinases. Protein kinases A (PKA) and G (PKG) phosphorylated CPI-17 at the inhibitory site (T38), but not PHI-1 (T57). Phosphorylated CPI-17 inhibited the activity of both the PP1 catalytic subunit (PP1c) and the myosin phosphatase holoenzyme (MPH) with IC(50) values of 1-8 nM. PKA predominantly phosphorylated a site distinct from the inhibitory T73 in KEPI, whereas PKG was ineffective. Integrin-linked kinase phosphorylated KEPI (T73) and this dramatically increased inhibition of PP1c (IC(50)=0.1 nM) and MPH (IC(50)=8 nM). These results suggest that the regulatory phosphorylation of CPI-17 and KEPI may involve distinct kinases and signaling pathways.