SARS-CoV-2 Mpro oligomerization as a potential target for therapy.

The main protease (Mpro) of SARS-CoV-2 is critical in the virus's replication cycle, facilitating the maturation of polyproteins into functional units. Due to its conservation across taxa, Mpro is a promising target for broad-spectrum antiviral drugs. Targeting Mpro with small molecule inhibitors, such as nirmatrelvir combined with ritonavir (Paxlovid™), which the FDA has approved for post-exposure treatment and prophylaxis, can effectively interrupt the replication process of the virus. A key aspect of Mpro's function is its ability to form a functional dimer. However, the mechanics of dimerization and its influence on proteolytic activity remain less understood. In this study, we utilized biochemical, structural, and molecular modelling approaches to explore Mpro dimerization. We evaluated critical residues, specifically Arg4 and Arg298, that are essential for dimerization. Our results show that changes in the oligomerization state of Mpro directly affect its enzymatic activity and dimerization propensity. We discovered a synergistic relationship influencing dimer formation, involving both intra- and intermolecular interactions. These findings highlight the potential for developing allosteric inhibitors targeting Mpro, offering promising new directions for therapeutic strategies.

The homeostatic function of Regnase-2 restricts neuroinflammation.

The precise physiological functions and mechanisms regulating RNase Regnase-2 (Reg-2/ZC3H12B/MCPIP2) activity remain enigmatic. We found that Reg-2 actively modulates neuroinflammation in nontransformed cells, including primary astrocytes. Downregulation of Reg-2 in these cells results in increased mRNA levels of proinflammatory cytokines IL-1ß and IL-6. In primary astrocytes, Reg-2 also regulates the mRNA level of Regnase-1 (Reg-1/ZC3H12A/MCPIP1). Reg-2 is expressed at high levels in the healthy brain, but its expression is reduced during neuroinflammation as well as glioblastoma progression. This process is associated with the upregulation of Reg-1. Conversely, overexpression of Reg-2 is accompanied by the downregulation of Reg-1 in glioma cells in a nucleolytic NYN/PIN domain-dependent manner. Interestingly, low levels of Reg-2 and high levels of Reg-1 correlate with poor-glioblastoma patients' prognoses. While Reg-2 restricts the basal levels of proinflammatory cytokines in resting astrocytes, its expression is reduced in IL-1ß-activated astrocytes. Following IL-1ß exposure, Reg-2 is phosphorylated, ubiquitinated, and degraded by proteasomes. Simultaneously, the Reg-2 transcript is destabilized by tristetraprolin (TTP) and Reg-1 through the AREs elements and conservative stem-loop structure present in its 3'UTR. Thus, the peer-control loop, of Reg-1 and Reg-2 opposing each other, exists. The involvement of TTP in Reg-2 mRNA turnover is confirmed by the observation that high TTP levels correlate with the downregulation of the Reg-2 expression in high-grade human gliomas. Additionally, obtained results reveal the importance of Reg-2 in inhibiting human and mouse glioma cell proliferation. Our current studies identify Reg-2 as a critical regulator of homeostasis in the brain.

Structural Determinants of Substrate Specificity of SplF Protease from Staphylococcus aureus.

Accumulating evidence suggests that six proteases encoded in the spl operon of a dangerous human pathogen, Staphylococcus aureus, may play a role in virulence. Interestingly, SplA, B, D, and E have complementary substrate specificities while SplF remains to be characterized in this regard. Here, we describe the prerequisites of a heterologous expression system for active SplF protease and characterize the enzyme in terms of substrate specificity and its structural determinants. Substrate specificity of SplF is comprehensively profiled using combinatorial libraries of peptide substrates demonstrating strict preference for long aliphatic sidechains at the P1 subsite and significant selectivity for aromatic residues at P3. The crystal structure of SplF was provided at 1.7 Å resolution to define the structural basis of substrate specificity of SplF. The obtained results were compared and contrasted with the characteristics of other Spl proteases determined to date to conclude that the spl operon encodes a unique extracellular proteolytic system.

On the mechanism of action of SJ-172550 in inhibiting the interaction of MDM4 and p53.

SJ-172550 (1) was previously discovered in a biochemical high throughput screen for inhibitors of the interaction of MDMX and p53 and characterized as a reversible inhibitor (J. Biol. Chem. 2010; 285:10786). Further study of the biochemical mode of action of 1 has shown that it acts through a complicated mechanism in which the compound forms a covalent but reversible complex with MDMX and locks MDMX into a conformation that is unable to bind p53. The relative stability of this complex is influenced by many factors including the reducing potential of the media, the presence of aggregates, and other factors that influence the conformational stability of the protein. This complex mechanism of action hinders the further development of compound 1 as a selective MDMX inhibitor.

Interaction of regulators Mdm2 and Mdmx with transcription factors p53, p63 and p73.

The negative regulation of p53, a major human tumor suppressor, by Mdm2 and Mdmx is crucial for the survival of a cell, whereas its aberrant function is a common feature of cancer.  Both Mdm proteins act through the spatial occlusion of the p53 transactivation (TA) domain and by the ubiquitination of p53, resulting in its degradation.  Two p53 homologues, p63 and p73, have been described in humans.  Unlike p53, these proteins regulate developmental processes rather than genome stability.  Both p63 and p73 contain TA domains homologous to that of p53, but relatively little is known about their regulation by Mdm2 or Mdmx.  Here, we present a detailed characterization of the interaction of Mdm2 and Mdmx with the TA domains of p63 and p73. Earlier reports of Mdm2 and Mdmx interactions with p73 are substantiated by the detailed quantitative characterization reported in this study. Most importantly, earlier contradictions concerning the presumed interaction of the Mdm proteins with p63 are convincingly resolved and for the first time, the affinities of these interactions are determined.  Finally, the contribution of these findings to our understanding of the physiological role of these interactions is discussed.

Electron paramagnetic resonance oxygen image hypoxic fraction plus radiation dose strongly correlates with tumor cure in FSa fibrosarcomas.

PURPOSE: Tumor hypoxia has long been known to produce resistance to radiation. In this study, electron paramagnetic resonance (EPR) oxygen imaging was investigated for its power to predict the success of tumor control according to tumor oxygenation level and radiation dose. METHODS AND MATERIALS: A total of 34 EPR oxygen images were obtained from the legs of C3H mice bearing 0.5-cm(3) FSa fibrosarcomas under both normal (air breathing) and clamped tumor conditions. Under the same conditions as those during which the images were obtained, the tumors were irradiated to a variety of doses near the FSa dose at which 50% of tumors were cured. Tumor tissue was distinguished from normal tissue using co-registration of the EPR oxygen images with spin-echo magnetic resonance imaging of the tumor and/or stereotactic localization. The tumor voxel statistics in the EPR oxygen image included the mean and median partial pressure of oxygen and the fraction of tumor voxels below the specified partial pressure of oxygen values of 3, 6, and 10 mm Hg. Bivariate logistic regression analysis using the radiation dose and each of the EPR oxygen image statistics to determine which best separated treatment failure from success. RESULTS: The measurements of the dose at which 50% of tumors were cured were similar to those found in published data for this syngeneic tumor. Bivariate analysis of 34 tumors demonstrated that tumor cure correlated with dose (p = 0.004) and with a <10 mm Hg hypoxic fraction (p = 0.023). CONCLUSION: Our results have shown that, together, radiation dose and EPR image hypoxic fraction separate the population of FSa fibrosarcomas that are cured from those that fail, thus predicting curability.

Enzymatic activity of the Staphylococcus aureus SplB serine protease is induced by substrates containing the sequence Trp-Glu-Leu-Gln.

Proteases are of significant importance for the virulence of Staphylococcus aureus. Nevertheless, their subset, the serine protease-like proteins, remains poorly characterized. Here presented is an investigation of SplB protease catalytic activity revealing that the enzyme possesses exquisite specificity and only cleaves efficiently after the sequence Trp-Glu-Leu-Gln. To understand the molecular basis for such selectivity, we solved the three-dimensional structure of SplB to 1.8 A. Modeling of substrate binding to the protease demonstrated that selectivity relies in part on a canonical specificity pockets-based mechanism. Significantly, the conformation of residues that ordinarily form the oxyanion hole, an essential structural element of the catalytic machinery of serine proteases, is not canonical in the SplB structure. We postulate that within SplB, the oxyanion hole is only formed upon docking of a substrate containing the consensus sequence motif. It is suggested that this unusual activation mechanism is used in parallel with classical determinants to further limit enzyme specificity. Finally, to guide future development, we attempt to point at likely physiological substrates and thus the role of SplB in staphylococcal physiology.

In vivo spin trapping of nitric oxide from animal tumors.

Spin trapping/electron paramagnetic resonance (EPR) spectroscopy allows specific detection of nitric oxide (NO) generation, in vivo. However, in order to detect an EPR signal in living organism, usually a stimulation of immune system with LPS is used to achieve higher than physiological NO levels. Here, we report non-invasive spin trapping of NO in tumors of non-treated, living animals. EPR spectroscopy was performed at S-band to detect NO in Cloudman S91 melanoma tumors growing in the tail of living, syngeneic hosts-DBA/2 mice. Iron (II) N-(dithiocarboxy)sarcosine Fe2+(DTCS)(2) was used as the spin trap. The results were confirmed by X-band ex vivo study. A characteristic three-line spectrum of NO-Fe(DTCS)(2) (A(N)=13 G) was observed (n=4, out of total n=6) in non-treated tumors and in tumors of animals treated with l-arginine. Substrate availability did not limit the detection of NO by spin trapping. Half-life time of the NO-Fe(DTCS)(2) in tumor tissue was about 60 min. The feasibility of non-invasive spin trapping/EPR spectroscopic detection of NO generated in tumor tissue in living animals, without additional activation of the immune system, was demonstrated for the first time.