Calpain-1 and calpain-2 are heterodimeric proteases consisting of a common small regulatory subunit CAPNS1 and a large catalytic subunit, CAPN1 or CAPN2, respectively. These calpains have emerged as potential therapeutic targets in cancer and other diseases through their roles in cell signaling pathways affecting sensitivity to chemotherapeutic and targeted drugs, and in promoting metastasis. While inhibition of calpains has the potential to provide therapeutic benefit to cancer patients, there are currently no clinically approved active site directed drugs that specifically and effectively inhibit them. However, the structures of calpain-1 and calpain-2 make them susceptible to allosteric inhibition aimed at interfering with heterodimerization of the catalytic and regulatory subunits, which is necessary for stability and proteolytic activity. Split-Nanoluciferase biosensors were generated to quantify the protein-protein interactions (PPIs) between the calcium-binding penta-EF hand (PEF) domains of CAPN1 or CAPN2 and CAPNS1. These biosensors were used to quantify the heterodimer dissociation constants (K_D) of calpain-1 and calpain-2, estimated at 185 nM and 509 nM, respectively, in the presence of 5 mM Ca²⁺; and 362 nM and 1651 nM, respectively, in the presence of Mg²⁺. The half-maximal Ca²⁺ concentrations supporting these PPIs for calpain-1 and calpain-2 were 59.9 μM and 940.8 μM, respectively. Molecular modeling, based on the crystal structure of calpain-2, was used to predict 20 residues of the PEF domains that contribute to heterodimerization. Individual point mutation of CAPNS1 at Q263 reduced the catalytic activity of calpain-2 to 51.0 ± 6.4 % in live cells.

In breast cancer, progression from localized stage I to distant metastatic stage IV disease is associated with a reduction of 5-year survival from nearly 100% to 23.2%. Expression of the calcium-activated protease isoforms calpain-1 and calpain-2 has been correlated with cell migration and invasion in vitro, metastatic potential in preclinical mouse models of cancer, and breast cancer prognosis in patients. It is unclear which of these two calpain isoforms is responsible for the apparent metastatic potential of cancer cells. Here, we demonstrate that while individual CRISPR-Cas9 knockouts of either CAPN1 or CAPN2 genes (encoding the catalytic subunits of calpain-1 and -2, respectively) reduce in vitro migration and marginally suppress in vivo metastasis, genetic disruption of both calpain-1 and calpain-2 through knockout of the CAPNS1 gene (encoding the common regulatory subunit of calpain-1 and -2) diminishes metastasis by 83.4 ± 13.6% in a mouse xenograft model of human triple-negative breast cancer. The effect of calpain-1/2 deficiency was replicated in vitro with a modified cell-permeable calpastatin (CAST)-based peptide inhibitor (cell migration reduced to 53.5 ± 11.0% of vehicle control). However, this peptide inhibitor was not effective in vivo at reducing metastasis under the conditions used (vehicle vs. CAST, 1.12 ± 1.35 lung metastases per mm² vs. 0.34 ± 0.20 metastases per mm²), likely due to rapid clearance, as indicated by the short serum half-life. This work demonstrates that calpain-1/2 disruption effectively abrogates metastasis and provides rationale for development of effective calpain inhibitors.

In this paper it is proposed a new method of symbolic system of linear algebraic equations (SSLAE) generation describing a parametric circuit in frequency

The D-trees method ensures near optimal factoring in the formed algebraic equations, which results in a significant cut-down of the time needed for their formation. The analysis of the example linear circuits with fixed and variable parameters presented in the paper revealed that application of the D-trees methods ensures from 10-to-100-fold time saving as compared to the standard MATLAB tools. Such reduction of time allows a considerable rise in the efficiency of the FS method in problems of statistical studies or optimization of electronic devices modelled by linear periodically time-varying circuits

Homeostatic immunoregulatory mechanisms that prevent adverse effects of immune overaction can serve as barriers to successful anticancer immunity, representing attractive targets to improve cancer immunotherapy. Here, we demonstrated the role of the nonreceptor tyrosine kinase Fes, abundantly expressed in immune cells, as an innate intracellular immune checkpoint. Host Fes deficiency delayed tumor onset in a gene dose-dependent manner and improved tumor control, survival, doxorubicin efficacy, and sensitized tumors to anti–PD-1 therapy in murine triple-negative breast cancer and melanoma models. These effects were associated with a shift to an antitumorigenic immune microenvironment. Fes-deficient macrophages displayed increased Toll-like receptor signaling, proinflammatory cytokine production, and antigen presentation to and activation of T cells, leading to increased cancer cell killing in vitro and tumor control in vivo. This study highlights Fes as an innate immune checkpoint with potential as a therapeutic target and a predictive biomarker to guide immune checkpoint inhibitor treatment. Significance: Fes activity modulates the inflammatory cytokine presentation and T-cell priming capabilities of macrophages, supporting the potential of Fes as a target for developing therapeutic and biomarker strategies to improve cancer immunotherapy.

In this paper, the time of solving such SSLAR was reduced by using one of subcircuit methods, namely, topological d-tree method. The existing d-tree method