UNASSIGNED: Cysteine proteases are involved in a broad range of biological functions, ranging from extracellular matrix turnover to immunity. Playing an important role in the onset and progression of several diseases, including cancer, immune-related and neurodegenerative disease, viral and parasitic infections, cysteine proteases represent an attractive drug target for the development of therapeutic tools.
UNASSIGNED: Recent scientific and patent literature focusing on the design and study of cysteine protease inhibitors with potential therapeutic application has been reviewed.
UNASSIGNED: The discovery of a number of effective structurally diverse cysteine protease inhibitors opened up new challenges and opportunities for the development of therapeutic tools. Mechanistic studies and the availability of X-ray crystal structures of some proteases, alone and in complex with inhibitors, provide crucial information for the rational design and development of efficient and selective cysteine protease inhibitors as preclinical candidates for the treatment of different diseases.

Phytocystatins are a type of proteinase inhibitor which are extensively studied for their specific inhibitory action against cysteine protease enzymes (CP) of insects and pathogens. Oryzacystatins (OC), a phytocystatin from rice inhibits CP in a reversible manner with its conserved tripartite wedge. OCs have important role in plant innate defense mechanism through phytohormonal signalling pathways. OC are induced in response to both biotic and abiotic stress conditions and are used to develop transgenic plants exhibiting resistance against stress conditions. In this review, we focus on the structure and mechanism of action of oryzacystatins, their possible role in plant physiology, biotic and abiotic stress tolerance mechanism in plants and their potential application strategies for future crop management studies.

The immunogenicity and efficacy of Fasciola DNA vaccines have not yet been comprehensively summarised in the form of a systematic review and meta-analysis. Though multiple vaccine studies with respect to Fasciola vaccines exist, the variance in the experimental parameters has made comparison difficult. We conducted a bibliographic database search in Scopus, PubMed, Science Direct, Cochrane Library, EMBASE and Web of Science databases, limited to publications from 1998 to 2017. The key words: Liver fluke, Fasciola hepatica, Fasciola gigantica, DNA vaccination, and immunogenicity were used in combination to form search strings. A total of 4760 studies were identified after initial screening, of which 14 qualified for systematic review and 7 for meta-analysis. The mean Odds Ratio (OR) for all studies was 0.565 (95% confidence interval (CI) of 0.293 to 1.087), which means the percentage of protection in terms of decreased fluke burden in animals vaccinated with DNA vaccines was 43.5%. A moderate protective efficacy was observed for cysteine protease and phosphoglycerate kinase vaccine antigen candidates (pooled OR and 95% CI, [0.542; 0.179-1.721] and [0.616; 0.219-1.735], respectively). Vaccine effectiveness was observed in individual studies and cohorts; however, the overall pooled efficacy for all vaccine candidates was found to be non-significant. Despite multiple individual studies showing promising results for various DNA vaccine candidates against fascioliasis, the pooled studies showed the non-significant effect of the vaccine formulations against fluke burden, and displayed minimal protective efficacy against Fasciola infection. Though promising results are observed in isolated studies, further animal trials with standardised experimental parameters are required to develop new vaccine candidates effective against Fasciola.

Cysteine proteases play multiple roles in basically all aspects of physiology and development. In plants, they are involved in growth and development and in accumulation and mobilization of storage proteins. Furthermore, they are engaged in signalling pathways and in the response to biotic and abiotic stresses. In animals and also in humans, they are responsible for senescence and apoptosis, prohormone processing, and ECM remodelling. When analyzed by zymography, the enzyme must be renaturated after SDS-PAGE. SDS must be washed out and substituted by Triton X-100. Gels are then further incubated under ideal conditions for activity detection. Cysteine proteases require an acidic pH (5.0-6.0) and a reducing agent, usually DTT. When screening biological samples, there is generally no previous clue on what peptidase class will be present, neither optimal proteolysis conditions are known. Hence, it is necessary to assess several parameters, such as incubation time, pH, temperature, influence of ions or reducing agents, and finally evaluate the inhibition profile. For detection of cysteine peptidase activity, the use of specific inhibitors, such as E-64, can be used to prevent the development of cysteine peptidase activity bands and positively confirm its presence. Here four different protocols to assess cysteine protease activity from different sources are presented.

Bromelain is a cysteine protease found in pineapple tissue. Because of its anti-inflammatory and anti-cancer activities, as well as its ability to induce apoptotic cell death, bromelain has proved useful in several therapeutic areas. The market for this protease is growing, and several studies exploring various properties of this molecule have been reported. This review aims to compile this data, and summarize the main findings on bromelain in the literature to date. The physicochemical properties and stability of bromelain under different conditions are discussed. Several studies on the purification of bromelain from crude extracts using a wide range of techniques such as liquid-liquid extractions by aqueous two-phase system, ultrafiltration, precipitation, and chromatography, have been reported. Finally, the various applications of bromelain are presented. This review therefore covers the main properties of bromelain, aiming to provide an up-to-date compilation of the data reported on this enzyme.