Structure determinants defining the specificity of papain-like cysteine proteases.

Papain-like cysteine proteases are widely expressed enzymes that mostly regulate protein turnover in the acidic conditions of lysosomes. However, in the last twenty years, these proteases have been evidenced to exert specific functions within different organelles as well as outside the cell. The most studied proteases of this family are human cysteine cathepsins involved both in physiological and pathological processes. The specificity of each protease to its substrates is mostly defined by the structure of the binding cleft. Different patterns of amino acid motif in this area determine the interaction between the protease and the ligands. Moreover, this specificity can be altered under the specific media conditions and in case other proteins are present. Understanding how this network works would allow researchers to design the diagnostic selective probes and therapeutic inhibitors. Moreover, this knowledge might serve as a key for redesigning and de novo engineering of the proteases for a wide range of applications.

Cathepsin D-Managing the Delicate Balance.

Lysosomal proteases play a crucial role in maintaining cell homeostasis. Human cathepsin D manages protein turnover degrading misfolded and aggregated proteins and favors apoptosis in the case of proteostasis disruption. However, when cathepsin D regulation is affected, it can contribute to numerous disorders. The down-regulation of human cathepsin D is associated with neurodegenerative disorders, such as neuronal ceroid lipofuscinosis. On the other hand, its excessive levels outside lysosomes and the cell membrane lead to tumor growth, migration, invasion and angiogenesis. Therefore, targeting cathepsin D could provide significant diagnostic benefits and new avenues of therapy. Herein, we provide a brief overview of cathepsin D structure, regulation, function, and its role in the progression of many diseases and the therapeutic potentialities of natural and synthetic inhibitors and activators of this protease.

Unravelling the Network of Nuclear Matrix Metalloproteinases for Targeted Drug Design.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that are responsible for the degradation of a wide range of extracellular matrix proteins, which are involved in many cellular processes to ensure the normal development of tissues and organs. Overexpression of MMPs has been observed to facilitate cellular growth, migration, and metastasis of tumor cells during cancer progression. A growing number of these proteins are being found to exist in the nuclei of both healthy and tumor cells, thus highlighting their localization as having a genuine purpose in cellular homeostasis. The mechanism underlying nuclear transport and the effects of MMP nuclear translocation have not yet been fully elucidated. To date, nuclear MMPs appear to have a unique impact on cellular apoptosis and gene regulation, which can have effects on immune response and tumor progression, and thus present themselves as potential therapeutic targets in certain types of cancer or disease. Herein, we highlight and evaluate what progress has been made in this area of research, which clearly has some value as a specific and unique way of targeting the activity of nuclear matrix metalloproteinases within various cell types.

Papain-Like Proteases as Coronaviral Drug Targets: Current Inhibitors, Opportunities, and Limitations.

Papain-like proteases (PLpro) of coronaviruses (CoVs) support viral reproduction and suppress the immune response of the host, which makes CoV PLpro perspective pharmaceutical targets. Their inhibition could both prevent viral replication and boost the immune system of the host, leading to the speedy recovery of the patient. Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the third CoV outbreak in the last 20 years. Frequent mutations of the viral genome likely lead to the emergence of more CoVs. Inhibitors for CoV PLpro can be broad-spectrum and can diminish present and prevent future CoV outbreaks as PLpro from different CoVs have conservative structures. Several inhibitors have been developed to withstand SARS-CoV and Middle East respiratory syndrome CoV (MERS-CoV). This review summarizes the structural features of CoV PLpro, the inhibitors that have been identified over the last 20 years, and the compounds that have the potential to become novel effective therapeutics against CoVs in the near future.

Redox-Mediated Post-Translational Modifications of Proteolytic Enzymes and Their Role in Protease Functioning.

Proteolytic enzymes play a crucial role in metabolic processes, providing the cell with amino acids through the hydrolysis of multiple endogenous and exogenous proteins. In addition to this function, proteases are involved in numerous protein cascades to maintain cellular and extracellular homeostasis. The redox regulation of proteolysis provides a flexible dose-dependent mechanism for proteolytic activity control. The excessive reactive oxygen species (ROS) and reactive nitrogen species (RNS) in living organisms indicate pathological conditions, so redox-sensitive proteases can swiftly induce pro-survival responses or regulated cell death (RCD). At the same time, severe protein oxidation can lead to the dysregulation of proteolysis, which induces either protein aggregation or superfluous protein hydrolysis. Therefore, oxidative stress contributes to the onset of age-related dysfunction. In the present review, we consider the post-translational modifications (PTMs) of proteolytic enzymes and their impact on homeostasis.

Novel applications of modification of thiol enzymes and redox-regulated proteins using S-methyl methanethiosulfonate (MMTS).

S-Methyl methanethiosulfonate (MMTS) is used in experimental biochemistry for alkylating thiol groups of protein cysteines. Its applications include mainly trapping of natural thiol-disulfide states of redox-sensitive proteins and proteins which have undergone S-nitrosylation. The reagent can also be employed as an inhibitor of enzymatic activity, since nucleophilic cysteine thiolates are commonly present at active sites of various enzymes. The advantage of using MMTS for this purpose is the reversibility of the formation of methylthio mixed disulfides, compared to irreversible alkylation using conventional agents. Additional benefits include good accessibility of MMTS to buried protein cysteines due to its small size and the simplicity of the protection and deprotection procedures. In this study we report examples of MMTS application in experiments involving oxidoreductase (glyceraldehyde-3-phosphate dehydrogenase, GAPDH), redox-regulated protein (recoverin) and cysteine protease (triticain-α). We demonstrate that on the one hand MMTS can modify functional cysteines in the thiol enzyme GAPDH, thereby preventing thiol oxidation and reversibly inhibiting the enzyme, while on the other hand it can protect the redox-sensitive thiol group of recoverin from oxidation and such modification produces no impact on the activity of the protein. Furthermore, using the example of the papain-like enzyme triticain-α, we report a novel application of MMTS as a protector of the primary structure of active cysteine protease during long-term purification and refolding procedures. Based on the data, we propose new lines of MMTS employment in research, pharmaceuticals and biotechnology for reversible switching off of undesirable activity and antioxidant protection of proteins with functional thiol groups.

Trends and Prospects of Plant Proteases in Therapeutics.

The main function of proteases in any living organism is the cleavage of proteins resulting in the degradation of damaged, misfolded and potentially harmful proteins and therefore providing the cell with amino acids essential for the synthesis of new proteins. Besides this main function, proteases may play an important role as signal molecules and participate in numerous protein cascades to maintain the vital processes of an organism. Plant proteases are no exception to this rule. Moreover, in contrast to humanencoded enzymes, many plant proteases possess exceptional features such as higher stability, unique substrate specificity and a wide pH range for enzymatic activity. These valuable features make plant-derived proteolytic enzymes suitable for many biomedical applications, and furthermore, the plants can serve as factories for protein production. Plant proteases are already applied in the treatment of several pathological conditions in the human organism. Some of the enzymes possess antitumour, antibacterial and antifungal activity. The collagenolytic activity of plant proteases determines important medical applications such as the healing of wounds and burn debridement. Plant proteases may affect blood coagulation processes and can be applied in the treatment of digestive disorders. The present review summarizes recent advances and possible applications for plant proteases in biomedicine, and proposes further development of plant-derived proteolytic enzymes in the biotechnology and pharmaceutical industries.

Rational Design of Recombinant Papain-Like Cysteine Protease: Optimal Domain Structure and Expression Conditions for Wheat-Derived Enzyme Triticain-α.

Triticain-α is a papain-like cysteine protease from wheat (Triticumaestivum L.) that possesses activity towards toxic gluten-derived peptides, and was thus proposed as a novel therapeutic tool for celiac disease. We report an original approach employing rational design of domain architecture of Triticain-α and selection of the appropriate expression system for development of cheap and efficient protocol yielding active recombinant enzyme. The segregated catalytic domain of Triticain-α did not adopt native structure in bacteria, neither being expressed as a single protein nor upon conjugation or co-expression with extrinsic chaperones. Meanwhile, its attachment to prodomain of the enzyme resulted in generation of insoluble (inclusion bodies) product that can be transformed into active protease upon refolding in vitro. The estimated yield of the product was affected by affinity six-histidine tag required for its single-step purification with the preferable N-terminal position of the tag. Expression of the two-domain Triticain-α construct in yeast (Pichiapastoris) strain GS115 and bacterial (Escherichia coli) strain Rosetta gami B (DE3) led to the accumulation of a soluble protein, which underwent autocatalytic maturation during expression (in yeast)/purification (in bacteria) procedures and exhibited pronounced protease activity. Furthermore, expression and solubility of such construct in Rosetta gami B (DE3) cells was improved by reducing the temperature of the bacterial growth yielding more active enzyme than yeast counterpart presumably due to facilitated formation of a characteristic disulfide bond critical for maintaining the catalytic site. We suggest that these findings are helpful for obtaining active Triticain-α preparations for scientific or medical applications, and can be employed for the design and production of beneficial recombinant products based on other papain-like cysteine proteases.