Myotis davidii cystatin A (MdCSTA), a stefin A-like from the Chinese native bat species M. davidii, was expressed as a recombinant protein and functionally characterized as a strong inhibitor of the cysteine proteases papain, human cathepsins L and B and the tick cathepsin L-like BmCL1. Despite the highly conserved amino acid sequences among stefins A from different vertebrates, MdCSTA presents a Methionine-2 residue at the N-terminal region and the second binding loop (pos 73-79) that differs from human stefin A (HsCSTA) and might be related to the lower inhibition constant (Ki) value presented by this inhibitor in comparison to human stefin A inhibition to cathepsin B. Therefore, to investigate the importance of these variable regions in cathepsin B inhibition, recombinant stefins A MdCSTA and HsCSTA containing mutations at the second amino acid residue and second binding loop were expressed and evaluated in kinetic assays. Enzymatic inhibition assays with cathepsin B revealed that switching the amino acid residues at position 2 and second binding loop region between bat and human CSTAs improved the HsCSTA\'s and reduced MdCSTA\'s inhibitory activity. Additionally, molecular docking analysis estimated lower energy values for the complex between MdCSTA-cathepsin B, in comparison to human CSTA-cathepsin B, while the mutants presented intermediate values, suggesting that other regions might contribute to the higher inhibitory activity against cathepsin B by MdCSTA. In conclusion, MdCSTA, the first bat\'s stefin A-like inhibitor to be functionally characterized, presented a higher inhibitory activity against cathepsin B in comparison to the human inhibitor, which is partially related to the glutamine-rich second binding loop and Met-2. Further structural analysis should be performed to elucidate potential inhibitor effects on cysteine proteinases.

The three mammalian TET dioxygenases oxidize the methyl group of 5-methylcytosine in DNA, and the oxidized methylcytosines are essential intermediates in all known pathways of DNA demethylation. To define the in vivo consequences of complete TET deficiency, we inducibly deleted all three Tet genes in the mouse genome. Tet1/2/3-inducible TKO (iTKO) mice succumbed to acute myeloid leukemia (AML) by 4 to 5 wk. Single-cell RNA sequencing of Tet iTKO bone marrow cells revealed the appearance of new myeloid cell populations characterized by a striking increase in expression of all members of the stefin/cystatin gene cluster on mouse chromosome 16. In patients with AML, high stefin/cystatin gene expression correlates with poor clinical outcomes. Increased expression of the clustered stefin/cystatin genes was associated with a heterochromatin-to-euchromatin compartment switch with readthrough transcription downstream of the clustered stefin/cystatin genes as well as other highly expressed genes, but only minor changes in DNA methylation. Our data highlight roles for TET enzymes that are distinct from their established function in DNA demethylation and instead involve increased transcriptional readthrough and changes in three-dimensional genome organization.

Cystatin proteins are known to form a superfamily of cysteine protease inhibitors, which play a key role in protein degradation and are related to different physiological processes, such as development and immunity. Currently, numerous immunoregulatory proteins, such as cystatins, are being used in the control and prevention of diseases in aquaculture. Thus, the objective of this study was to produce recombinant cystatin (rCYST-B) from the red piranha Pygocentrus nattereri and to evaluate its effect on bacterial growth. The gene that encodes cystatin-B was isolated from the spleen of P. nattereri and cloned in an expression system. The protein was produced via a heterologous system involving the yeast Pichia pastoris X-33. The inhibitory activity of purified cystatin-B was evaluated on papain using different concentrations (0-80.0 μg/μL) of rCYST-B. The bacteriostatic action of the protein was evaluated using the Kirby-Bauer method on the growth of Escherichia coli and Bacillus subtilis. rCYST-B showed 100% inhibition at a concentration of 60 μg/μL. Moreover, the bacteriostatic activity of E. coli and B. subtilis showed inhibition of 40.36 and 49.08% compared to the negative control (phosphate buffer), respectively. These results suggest that recombinant CYST-B has biotechnological potential for use in aquaculture.