Covalent Immobilization of Thiol Proteinases on Chitosan

Plant enzymes such as ficin (EC 3.4.22.3), papain (EC 3.4.22.2) and bromelain (EC 3.4.22.4) are obtained from tropical plants. These biocatalysts belong to thiol proteases, in the active center of which cysteine is contained. Ficin, papain and bromelain have a wide substrate specificity, which provides a demand for their use in various industries. Enzymes in the free state are less commonly used; immobilized biocatalysts are the preferred form. The aim of this work was to determine the optimal concentration of a crosslinking agent in the covalent immobilization of ficin, papain and bromelain on a chitosan matrix. Ficin, papain and bromelain (Sigma) were chosen as objects of study. An acid-soluble chitosan (350 kDa, Bioprogress CJSC) was used as an immobilization carrier. The concentration range of glutaraldehyde (crosslinking agent) ranged from 1 to 25%. Suitable concentrations of glutaraldehyde for covalent immobilization were identified by the optimal ratio of protein content (mg per g of carrier), total activity (in units per ml of solution) and specific activity (in units per mg of protein). It was shown that for covalent immobilization of ficin and bromelain on a chitosan matrix, it is most promising to use 10% glutaraldehyde. For immobilization of papain on chitosan by covalent means, the concentration of glutaraldehyde equal to 20% is optimal.

Targeted Oxidation Strategy (TOS) for Potential Inhibition of Coronaviruses by Disulfiram — a 70-Year Old Anti-Alcoholism Drug

<p>In the new millennium, the outbreak of new coronavirus has happened three times: SARS-CoV, MERS-CoV, and 2019-nCoV. Unfortunately, we still have no pharmaceutical weapons against the diseases caused by these viruses. The pandemic of 2019-nCoV reminds us of the urgency to search new drugs with totally different mechanism that may target the weaknesses specific to coronaviruses. Herein, we disclose a new targeted oxidation strategy (TOS II) leveraging non-covalent interactions potentially to oxidize and inhibit the activities of cytosolic thiol proteins via thiol/thiolate oxidation to disulfide (TOD). Quantum mechanical calculations show encouraging results supporting the feasibility to selectively oxidize thiol of targeted proteins via TOS II even in relatively reducing cytosolic microenvironments. Molecular docking against the two thiol proteases M^pro and PL^pro of 2019-nCoV provide evidence to support a TOS II mechanism for two experimentally identified anti-2019-nCoV disulfide oxidants: disulfiram and PX-12. Remarkably, disulfiram is an anti-alcoholism drug approved by FDA 70 years ago, thus it can be immediately used in phase III clinical trial for anti-2019-nCoV treatment. Finally, a preliminary list of promising TOS II drug candidates targeting the two thiol proteases of 2019-nCoV are proposed upon virtual screening of 32143 disulfides.</p>

Targeted Oxidation Strategy (TOS) for Potential Inhibition of Coronaviruses by Disulfiram — a 70-Year Old Anti-Alcoholism Drug

<p>In the new millennium, the outbreak of new coronavirus has happened three times: SARS-CoV, MERS-CoV, and 2019-nCoV. Unfortunately, we still have no pharmaceutical weapons against the diseases caused by these viruses. The pandemic of 2019-nCoV reminds us of the urgency to search new drugs with totally different mechanism that may target the weaknesses specific to coronaviruses. Herein, we disclose a new targeted oxidation strategy (TOS II) leveraging non-covalent interactions potentially to oxidize and inhibit the activities of cytosolic thiol proteins via thiol/thiolate oxidation to disulfide (TOD). Quantum mechanical calculations show encouraging results supporting the feasibility to selectively oxidize thiol of targeted proteins via TOS II even in relatively reducing cytosolic microenvironments. Molecular docking against the two thiol proteases M^pro and PL^pro of 2019-nCoV provide evidence to support a TOS II mechanism for two experimentally identified anti-2019-nCoV disulfide oxidants: disulfiram and PX-12. Remarkably, disulfiram is an anti-alcoholism drug approved by FDA 70 years ago, thus it can be immediately used in phase III clinical trial for anti-2019-nCoV treatment. Finally, a preliminary list of promising TOS II drug candidates targeting the two thiol proteases of 2019-nCoV are proposed upon virtual screening of 32143 disulfides.</p>

Fission yeast Cyk3p is a transglutaminase-like protein that participates in cytokinesis and cell morphogenesis

Cell morphogenesis is a complex process that relies on a diverse array of proteins and pathways. We have identified a transglutaminase-like protein (Cyk3p) that functions in fission yeast morphogenesis. The phenotype of a cyk3 knockout strain indicates a primary role for Cyk3p in cytokinesis. Correspondingly, Cyk3p localizes both to the actomyosin contractile ring and the division septum, promoting ring constriction, septation, and subsequent cell separation following ring disassembly. In addition, Cyk3p localizes to polarized growth sites and plays a role in cell shape determination, and it also appears to contribute to cell integrity during stationary phase, given its accumulation as dynamic puncta at the cortex of such cells. Our results and the conservation of Cyk3p across fungi point to a role in cell wall synthesis and remodeling. Cyk3p possesses a transglutaminase domain that is essential for function, even though it lacks the catalytic active site. In a wider sense, our work illustrates the physiological importance of inactive members of the transglutaminase family, which are found throughout eukaryotes. We suggest that the proposed evolution of animal transglutaminase cross-linking activity from ancestral bacterial thiol proteases was accompanied by the emergence of a subclass whose function does not depend on enzymatic activity.

Multifunctional role of plant cysteine proteinases.

Cysteine proteinases also referred to as thiol proteases play an essential role in plant growth and development but also in senescence and programmed cell death, in accumulation of storage proteins such as in seeds, but also in storage protein mobilization. Thus, they participate in both anabolic and catabolic processes. In addition, they are involved in signalling pathways and in the response to biotic and abiotic stresses. In this review an attempt was undertaken to illustrate these multiple roles of cysteine proteinases and the mechanisms underlying their action.