Unveiling the Roles of Cysteine Proteinases F and W: From Structure to Pathological Implications and Therapeutic Targets.

Cysteine cathepsins F and W are members of the papain-like cysteine protease family, which have distinct structural features and functional roles in various physiological and pathological processes. This review provides a comprehensive overview of the current understanding of the structure, biological functions, and pathological implications of cathepsins F and W. Beginning with an introduction to these proteases, we delve into their structural characteristics and elucidate their unique features that dictate their enzymatic activities and substrate specificity. We also explore the intricate involvement of cathepsins F and W in malignancies, highlighting their role as potential biomarkers and therapeutic targets in cancer progression. Furthermore, we discuss the emerging roles of these enzymes in immune response modulation and neurological disorders, shedding light on their implications in autoimmune and neurodegenerative diseases. Finally, we review the landscape of inhibitors targeting these proteases, highlighting their therapeutic potential and challenges in clinical translation. This review brings together the diverse facets of cysteine cathepsins F and W, providing insights into their roles in health and disease and guiding future investigations for therapeutic advances.

Evaluation of molecular mechanisms of riboflavin anti-COVID-19 action reveals anti-inflammatory efficacy rather than antiviral activity.

BACKGROUND: Riboflavin (vitamin B2) is one of the most important water-soluble vitamins and a coenzyme involved in many biochemical processes. It has previously been shown that adjuvant therapy with flavin mononucleotide (a water-soluble form of riboflavin) correlates with normalization of clinically relevant immune markers in patients with COVID-19, but the mechanism of this effect remains unclear. Here, the antiviral and anti-inflammatory effects of riboflavin were investigated to elucidate the molecular mechanisms underlying the riboflavin-induced effects. METHODS: Riboflavin was evaluated for recombinant SARS-CoV-2 PLpro inhibition in an enzyme kinetic assay and for direct inhibition of SARS-CoV-2 replication in Vero E6 cells, as well as for anti-inflammatory activity in polysaccharide-induced inflammation models, including endothelial cells in vitro and acute lung inflammation in vivo. RESULTS: For the first time, the ability of riboflavin at high concentrations (above 50 µM) to inhibit SARS-CoV-2 PLpro protease in vitro was demonstrated; however, no inhibition of viral replication in Vero E6 cells in vitro was found. At the same time, riboflavin exerted a pronounced anti-inflammatory effect in the polysaccharide-induced inflammation model, both in vitro, preventing polysaccharide-induced cell death, and in vivo, reducing inflammatory markers (IL-1ß, IL-6, and TNF-α) and normalizing lung histology. CONCLUSIONS: It is concluded that riboflavin reveals anti-inflammatory rather than antiviral activity for SARS-CoV-2 infection. GENERAL SIGNIFICANCE: Riboflavin could be suggested as a promising compound for the therapy of inflammatory diseases of broad origin.

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.

Questionnaire-based survey to determine an at-risk group for coeliac disease among school children in Moscow, Russia.

AIM: Coeliac disease (CD) is a chronic digestive disorder which presents in diverse ways and is under-diagnosed. The purpose of this study was to provide insights into suspected CD among Russian schoolchildren, through defining the percentage of participants in an 'at-risk' group for CD in a paediatric cohort, by means of a questionnaire as a primary screening tool. METHODS: Russian school children of both sexes age 7-18 years were enrolled in a population-based study to identify individuals affected by CD. Each participant was presented with a structured questionnaire based on criteria that can be used to reveal symptomatic signs of CD. Following on, we developed a case-finding strategy for the 'at-risk' group, based on serological and genetic testing and, where possible, endoscopic examination of participants. RESULTS: 10.2% of questionnaire respondents (312/3070) were classified as an at-risk group. Pathobiological CD analysis of this group returned positive test results for 13.5% of participants (42/312), and 0.6% of them (2/312) had CD confirmed by biopsy sample analysis. CONCLUSIONS: Our findings suggest that at-risk groups among children with symptomatic or some oligosymptomatic CD presentations can be identified through adopting a questionnaire as part of a population-based screening survey, if generally accepted screening programs are inaccessible.

Clinical Combinatorial Treatments Based on Cancer Vaccines: Combination with Checkpoint Inhibitors and Beyond.

The efficacy of the cancer vaccine is influenced by several factors, but one of the most important is the immunosuppressive tumor microenvironment, which can attenuate treatment effects. The combination of therapeutic cancer vaccines with other immunotherapies or conventional therapeutic approaches can promote vaccine efficacy by increasing immune surveillance and tumor immunogenicity and modulating immune escape in the tumor microenvironment. Inhibitory checkpoints have a significant role in the modulation of anticancer immune responses, and according to preclinical and clinical trials, administration of immune checkpoint inhibitors (ICIs) in combination with cancer vaccines can markedly improve their therapeutic effects, considering their low clinical efficacy. In addition, these combinatorial therapies have acceptable safety and minimal additional toxicity compared to single-agent cancer vaccines or ICIs. In this review, based on the results of previous studies, we introduce and discuss treatments that can be combined with therapeutic cancer vaccines to improve their potency. Our major focus is on checkpoint blockade therapies, which are the most well-known and applicable immunotherapies.

Cancer Vaccine in Cold Tumors: Clinical Landscape, Challenges, and Opportunities.

The idea of cancer immunotherapy is to stimulate the immune system to fight tumors without destroying normal cells. One of the anticancer therapy methods, among many, is based on the use of cancer vaccines that contain tumor antigens in order to induce immune responses against tumors. However, clinical trials have shown that the use of such vaccines as monotherapy is ineffective in many cases since they do not cause a strong immune response. Particular tumors are resistant to immunotherapy due to the absence or insufficient infiltration of tumors with CD8+ T cells, and hence, they are called cold or non-inflamed tumors. Cold tumors are characterized by a lack of CD8+ T cell infiltration, the presence of anti-inflammatory myeloid cells, tumor-associated M2 macrophages, and regulatory T cells. It is very important to determine the stage of the antitumor response that does not work properly in order to use the right strategy. Applying other therapeutic methods alongside cancer vaccines can be more rational for cold tumors, which do not provoke the immune system strongly. Herein, we indicate some combinational therapies that have been used or are in progress for cold tumor treatment alongside vaccines.

Nanomedicine for increasing the oral bioavailability of cancer treatments.

Oral administration is an appealing route of delivering cancer treatments. However, the gastrointestinal tract is characterized by specific and efficient physical, chemical, and biological barriers that decrease the bioavailability of medications, including chemotherapeutics. In recent decades, the fields of material science and nanomedicine have generated several delivery platforms with high potential for overcoming multiple barriers associated to oral administration. This review describes the properties of several nanodelivery systems that improve the bioavailability of orally administered therapeutics, highlighting their advantages and disadvantages in generating successful anticancer oral nanomedicines.

Cathepsin S Cleaves BAX as a Novel and Therapeutically Important Regulatory Mechanism for Apoptosis.

Certain lysosomal cathepsin proteins have come into focus as being good candidates for therapeutic targeting, based on them being over-expressed in a variety of cancers and based on their regulation of the apoptotic pathway. Here, we report novel findings that highlight the ability of cathepsin S expression to be up-regulated under Paclitaxel-stimulatory conditions in kidney cell lines and it being able to cleave the apoptotic p21 BAX protein in intact cells and in vitro. Consistent with this, we demonstrate that this effect can be abrogated in vitro and in mammalian cells under conditions that utilize dominant-inhibitory cathepsin S expression, cathepsin S expression-knockdown and through the activity of a novel peptide inhibitor, CS-PEP1. Moreover, we report a unique role for cathepsin S in that it can cleave a polyubiquitinated-BAX protein intermediate and is a step that may contribute to down-regulating post-translationally-modified levels of BAX protein. Finally, CS-PEP1 may possess promising activity as a potential anti-cancer therapeutic against chemotherapeutic-resistant Renal Clear Cell Carcinoma kidney cancer cells and for combined uses with therapeutics such as Paclitaxel.

Intrinsically Connected: Therapeutically Targeting the Cathepsin Proteases and the Bcl-2 Family of Protein Substrates as Co-regulators of Apoptosis.

Taken with the growing importance of cathepsin-mediated substrate proteolysis in tumor biology and progression, the focus and emphasis placed on therapeutic design and development is coming into fruition. Underpinning this approach is the invariable progression from the direction of fully characterizing cathepsin protease members and their substrate targets, towards targeting such an interaction with tangible therapeutics. The two groups of such substrates that have gained much attention over the years are the pro- and anti- apoptotic protein intermediates from the extrinsic and intrinsic signaling arms of the apoptosis pathway. As proteins that are central to determining cellular fate, some of them present themselves as very favorable candidates for therapeutic targeting. However, considering that both anti- and pro- apoptotic signaling intermediates have been reported to be downstream substrates for certain activated cathepsin proteases, therapeutic targeting approaches based on greater selectivity do need to be given greater consideration. Herein, we review the relationships shared by the cathepsin proteases and the Bcl-2 homology domain proteins, in the context of how the topical approach of adopting 'BH3-mimetics' can be explored further in modulating the relationship between the anti- and pro- apoptotic signaling intermediates from the intrinsic apoptosis pathway and their upstream cathepsin protease regulators. Based on this, we highlight important future considerations for improved therapeutic design.

Current Status and Perspectives of Protease Inhibitors and Their Combination with Nanosized Drug Delivery Systems for Targeted Cancer Therapy.

In cancer treatments, many natural and synthetic products have been examined; among them, protease inhibitors are promising candidates for anti-cancer agents. Since dysregulated proteolytic activities can contribute to tumor development and metastasis, antagonization of proteases with tailored inhibitors is an encouraging approach. Although adverse effects of early designs of these inhibitors disappeared after the introduction of next-generation agents, most of the proposed inhibitors did not pass the early stages of clinical trials due to their nonspecific toxicity and lack of pharmacological effects. Therefore, new applications that modulate proteases more specifically and serve their programmed way of administration are highly appreciated. In this context, nanosized drug delivery systems have attracted much attention because preliminary studies have demonstrated that the therapeutic capacity of inhibitors has been improved significantly with encapsulated formulation as compared to their free forms. Here, we address this issue and discuss the current application and future clinical prospects of this potential combination towards targeted protease-based cancer therapy.

Making Connections: p53 and the Cathepsin Proteases as Co-Regulators of Cancer and Apoptosis.

While viewed as the "guardian of the genome", the importance of the tumor suppressor p53 protein has increasingly gained ever more recognition in modulating additional modes of action related to cell death. Slowly but surely, its importance has evolved from a mutated genetic locus heavily implicated in a wide array of cancer types to modulating lysosomal-mediated cell death either directly or indirectly through the transcriptional regulation of the key signal transduction pathway intermediates involved in this. As an important step in determining the fate of cells in response to cytotoxicity or during stress response, lysosomal-mediated cell death has also become strongly interwoven with the key components that give the lysosome functionality in the form of the cathepsin proteases. While a number of articles have been published highlighting the independent input of p53 or cathepsins to cellular homeostasis and disease progression, one key area that warrants further focus is the regulatory relationship that p53 and its isoforms share with such proteases in regulating lysosomal-mediated cell death. Herein, we review recent developments that have shaped this relationship and highlight key areas that need further exploration to aid novel therapeutic design and intervention strategies.

Dendritic Cells in Anticancer Vaccination: Rationale for Ex Vivo Loading or In Vivo Targeting.

Dendritic cells (DCs) have shown great potential as a component or target in the landscape of cancer immunotherapy. Different in vivo and ex vivo strategies of DC vaccine generation with different outcomes have been proposed. Numerous clinical trials have demonstrated their efficacy and safety in cancer patients. However, there is no consensus regarding which DC-based vaccine generation method is preferable. A problem of result comparison between trials in which different DC-loading or -targeting approaches have been applied remains. The employment of different DC generation and maturation methods, antigens and administration routes from trial to trial also limits the objective comparison of DC vaccines. In the present review, we discuss different methods of DC vaccine generation. We conclude that standardized trial designs, treatment settings and outcome assessment criteria will help to determine which DC vaccine generation approach should be applied in certain cancer cases. This will result in a reduction in alternatives in the selection of preferable DC-based vaccine tactics in patient. Moreover, it has become clear that the application of a DC vaccine alone is not sufficient and combination immunotherapy with recent advances, such as immune checkpoint inhibitors, should be employed to achieve a better clinical response and outcome.

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.

Lost or Forgotten: The nuclear cathepsin protein isoforms in cancer.

While research into the role of cathepsins has been progressing at an exponential pace over the years, research into their respective isoform proteins has been less frenetic. In view of the functional and biological potential of such protein isoforms in model systems for cancer during their initial discovery, much later they have offered a new direction in the field of cathepsin basic and applied research. Consequently, the analysis of such isoforms has laid strong foundations in revealing other important regulatory aspects of the cathepsin proteins in general. In this review article, we address these key aspects of cathepsin isoform proteins, with particular emphasis on how they have shaped what is now known in the context of nuclear cathepsin localization and what potential these hold as nuclear-based therapeutic targets in cancer.

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.

Advances in the Development of Anticancer HSP-based Vaccines.

Current advances in cancer treatment are based on the recent discoveries of molecular mechanisms of tumour maintenance. It was shown that heat shock proteins (HSPs) play a crucial role in the development of immune response against tumours. Thus, HSPs represent multifunctional agents not only with chaperone functions, but also possessing immunomodulatory properties. These properties are exploited for the development of HSP-based anticancer vaccines aimed to induce cytotoxic responses against tumours. To date, a number of strategies have been suggested to facilitate HSP-based vaccine production and to increase its effectiveness. The present review focuses on the current trend for the development of HSPbased vaccines aimed at inducing strong immunological tumour-specific responses against cancer cells of distinct etiology and localization.

Autoantibody against arrestin-1 as a potential biomarker of renal cell carcinoma.

Renal cell carcinoma (RCC) is the second-most common uronephrological cancer. In the absence of specific symptoms, early diagnosis of RCC is challenging. Monitoring of the aberrant expression of tumour-associated antigens (TAAs) and related autoantibody response is considered as a novel approach of RCC diagnostics. The aim of this study was to examine the aberrant expression of arrestin-1 in renal tumours, to investigate the possible epigenetic mechanism underlying arrestin-1 expression, and to assess the frequency of anti-arrestin-1 autoantibody response. Immunohistochemistry was used to assess the presence of arrestin-1 in primary tumours and metastases of 39 patients with RCC and renal oncocytoma. Bisulfite sequencing was employed to analyse the methylation status of the promoter of the SAG gene encoding arrestin-1. Western blot analysis was performed to detect autoantibodies against arrestin-1 in serum samples of 36 RCC and oncocytoma patients. Arrestin-1 was found to be expressed in RCC (58.7% of cases) and renal oncocytoma (90% of cases) cells, while being absent in healthy kidney. The expression of arrestin-1 in RCC metastases was more prominent than in primary tumours. Hypomethylation of the SAG gene promoter is unlikely to be the mechanism for the aberrant expression of arrestin-1. Autoantibodies against arrestin-1 were detected in sera of 75% of RCC patients. Taken together, our findings suggest employment of autoantibody against arrestin-1 as biomarker of RCC.

Current Paediatric Coeliac Disease Screening Strategies and Relevance of Questionnaire Survey.

Coeliac disease (CD) is an autoimmune enteropathy triggered by the ingestion of gluten-containing grains in genetically predisposed individuals. Identification of CD in clinical practice is often difficult due to the manifestation of non-specific symptoms and signs, so a relatively significant proportion of CD cases remain undiagnosed. Timely detection of the disease is necessary to provide an appropriate approach to control of the disease treatment, in order to avoid potential complications. This is even more important in the case of children and adolescents, to ensure their proper growth and development. In this review, we discuss the data on the current strategies for CD detection among paediatric populations and the role of questionnaire-based discovery of CD cases in the area of interest. We assume that mass screening is a preferable strategy for finding CD cases within the paediatric population because this could uncover symptomatic, oligosymptomatic, and asymptomatic CD cases. However, under conditions of limited financial resources, screening for CD in risk groups, members of which can be identified using questionnaires, is essential. The pros and cons of CD screening in paediatric populations are presented. These depend on a number of situational criteria (cost-effectiveness, lack of awareness), but screening is designed to improve the detection of the disease and therefore improve the quality of life of patients.

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.