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Transmembrane serine protease 2 (TMPRSS2) is a membrane-bound protease belonging to the type II transmembrane serine protease (TTSP) family. It is a multidomain protein, including a serine protease domain responsible for its self-activation. The protein has been implicated as an oncogenic transcription factor and for its ability to cleave (prime) the SARS-CoV-2 spike protein. In order to characterize the TMPRSS2 biochemical properties, we expressed the serine protease domain (rTMPRSS2_SP) in Komagataella phaffii using the pPICZαA vector and purified it using immobilized metal affinity (Ni Sepharose™ excel) and size exclusion (Superdex 75) chromatography. We explored operational fluorescence resonance energy transfer FRET peptides as substrates. We chose the peptide Abz-QARK-(Dnp)-NH2 (Abz = ortho-aminobenzoic acid, the fluorescence donor, and Dnp = 2,4-dinitrophenyl, the quencher group) as a substrate to find the optimal conditions for maximum enzymatic activity. We found that metallic ions such as Ca2+ and Na+ increased enzymatic activity, but ionic surfactants and reducing agents decreased catalytic capacity. Finally, we determined the rTMPRSS2_SP stability for long-term storage. Altogether, our results represent the first comprehensive characterization of TMPRSS2's biochemical properties, providing valuable insights into its serine protease domain. [ABSTRACT FROM AUTHOR]

BACKGROUND: SET/I2PP2A is a multifunctional protein that is up-regulated in head and neck squamous cell carcinoma (HNSCC). The action of SET in HNSCC tumorigenicity is unknown. METHODS: Stable SET knockdown by shRNA (shSET) was established in three HNSCC cell lines: HN12, HN13, and Cal27. Protein expression and phosphorylated protein levels were determined by Western blotting and immunofluorescence, cell migration and invasion were measured by functional analysis, and PP2A activity was determined using a serine/threonine phosphatase assay. A real-time PCR array was used to quantify 84 genes associated with cell motility. Metalloproteinase (MMP) activity was assessed by zymographic and fluorometric assays. HN12shSET xenograft tumors (flank and tongue models) were established in Balb/c nude mice; the xenograft characteristics and cisplatin sensitivity were demonstrated by macroscopic, immunohistochemical, and histological analyses, as well as lymph node metastasis by histology. RESULTS: The HN12shSET cells displayed reduced ERK1/2 and p53 phosphorylation compared with control. ShSET reduced HN12 cell proliferation and increased the sub-G1 population of HN12 and Cal27 cells. Increased PP2A activity was also associated with shSET. The PCR array indicated up-regulation of three mRNAs in HN12 cells: vimentin, matrix metalloproteinase-9 (MMP9) and non-muscle myosin heavy chain IIB. Reduced E-cadherin and pan-cytokeratin, as well as increased vimentin, were also demonstrated as the result of SET knockdown. These changes were accompanied by an increase in MMP-9 and MMP-2 activities, migration and invasion. The HN12shSET subcutaneous xenograft tumors presented a poorly differentiated phenotype, reduced cell proliferation, and cisplatin sensitivity. An orthotopic xenograft tumor model using the HN12shSET cells displayed increased metastatic potential. CONCLUSIONS: SET accumulation has important actions in HNSCC. As an oncogene, SET promotes cell proliferation, survival, and resistance to cell death by cisplatin in vivo. As a metastasis suppressor, SET regulates invasion, the epithelial mesenchymal transition, and metastasis.

Water-insoluble exopolysaccharides (I-EPS) are a virulence factor for dental biofilms. It has already been demonstrated that mango pulp induces the secretion of glucan-hydrolytic enzymes in the fungus Trichoderma harzianum, and that they have an effect on I-EPS from young biofilms. Aim: Evaluate the effect of mango peel as an enzyme inducer in T. harzianum, and the effect of enzymes secreted on mature biofilms. Methods: Fractions of the peel (PL) and ethanol-precipitated pulp (PP) of Tommy Atkins mangoes were sterilized and added to a culture medium containing T. harzianum for induction of hydrolytic enzymes. After 192 h, the culture medium was centrifuged and the supernatant (enzyme extract) was used as treatment on S. mutans biofilms (n=9): a) NaCl 0.9 %; b) 0.12 % chlorhexidine digluconate; and c) extract of enzymes induced by PL or PP. Acidogenicity, bacterial viability, quantification of insoluble polysaccharides, and three-dimensional analysis of the biofilm by scanning electron microscopy (SEM) was performed. Data were analyzed by ANOVA followed by the Tukey test (α=5 %). Results: The hydrolytic enzymes did not alter the metabolism or bacterial viability of the biofilm (p<0.05). Although the images obtained by SEM suggest some degree of matrix degradation, the quantification of I-EPS for the PL and PP groups did not differ from the control group (p>0.05), suggesting a slight effect on the disorganization of the mature S. mutans biofilm. Conclusion: The results suggest that mango peel fraction can induce secretion of mutanase by T. harzianum, however in an insufficient amount to generate significant degradation on cariogenic biofilm. [ABSTRACT FROM AUTHOR]

Recently, there has been an increase in the demand for enzymes with modified activity, specificity, and stability. Enzyme engineering is an important tool to meet the demand for enzymes adjusted to different industrial processes. Knowledge of the structure and function of enzymes guides the choice of the best strategy for engineering enzymes. Each enzyme engineering strategy, such as rational design, directed evolution, and semi‐rational design, has specific applications, as well as limitations, which must be considered when choosing a suitable strategy. Engineered enzymes can be optimized for different industrial applications by choosing the appropriate strategy. This review features engineered enzymes that have been applied in food, animal feed, pharmaceuticals, medical applications, bioremediation, biofuels, and detergents. [ABSTRACT FROM AUTHOR]

The migratory behavior of freshwater shrimps may be affected by natural barriers in limnetic environments. This study evaluated the river areas separated by natural barriers, such as waterfalls, which affect the amphidromous shrimps' (Potimirim brasiliana) population features and reproductive aspects. Results indicate that in the Félix and Prumirim Rivers from southeastern Brazil shrimps show few differences in sampling areas, and these differences may not be causally related to the waterfalls. This is demonstrated by the absence of a pattern in the size and sex ratio in each area and the absence of a significant difference in most reproductive aspects. The presence of juveniles and reproductive individuals in all sampling areas strongly indicates a constant migration along them in both rivers, indicating that all individuals evaluated correspond to one single patchy population structure for each river. This migration conducted by P. brasiliana , such as its crawling behavior, demonstrated that it would be important to maintain the minimum number of individuals flowing between the different river sampling areas in this shrimp group. Thus, based on a helpful model observed in P. brasiliana , the results help us understand how natural barriers may affect the populations of amphidromous shrimp and how the migration behavior up- and downstream can help sustain the population. This premise can help future construction decisions and impacts of unnatural barriers, such as dams. [ABSTRACT FROM AUTHOR]