Activated MST2 kinase is free of kinetic regulation

Canonically, MST1/2 functions as a core kinase of the Hippo pathway and non-canonically is both activated during apoptotic signaling and acts in concert with RASSFs in T-cells. Faithful signal transduction relies on both appropriate activation and regulated substrate phosphorylation by the activated kinase. Considerable progress has been made understanding the molecular mechanisms regulating activation of MST1/2 and identifying downstream signaling events. Here we present a kinetic analysis analyzing how the ability of MST1/2 to phosphorylate substrates is regulated. Using a steady state kinetic system, we parse the contribution of different factors including the domains of MST2, phosphorylation, caspase cleavage, and complex formation to MST2 activity. In the unphosphorylated state, we find the SARAH domain stabilizes substrate binding. Phosphorylation, we also determine, drives activation of MST2 and that once activated the kinase domain is free of regulation. The binding partners SAV1, MOB1A, and RASSF5 do not alter the kinetics of phosphorylated MST2. We also show that the caspase cleaved MST2 fragment is as active as full-length suggesting that the linker region of MST2 does not inhibit the catalytic activity of the kinase domain but instead regulates MST2 activity through non-catalytic mechanisms. This kinetic analysis helps establish a framework for interpreting how signaling events, mutations, and post-translational modifications contribute to signaling of MST2 in vivo.

KPC-39-mediated resistance to Ceftazidime-Avibactam in a Klebsiella pneumoniae ST307 clinical isolate

Resistance to ceftazidime–avibactam (CAZ-AVI) combination is being increasingly reported. Here, we report a CAZ-AVI resistant Klebsiella pneumoniae belonging to the high-risk ST307 clone and producing KPC-39, a single amino-acid variant of KPC-3 (A172T). Cloning experiments, steady state kinetic parameters and molecular dynamics simulations revealed a loss of carbapenemase activity and an increased affinity for ceftazidime. KPC-39 was identified in a patient without prior exposure to CAZ-AVI, suggesting silent dissemination in European healthcare settings.

Detection and characterization of VIM-52, a new variant of VIM-1 from Klebsiella pneumoniae clinical isolate

Over the last two decades, antimicrobial resistance has become a global health problem. In Gram-negative bacteria, metallo-β-lactamases (MBLs), which inactivate virtually all β-lactams, increasingly contribute to this phenomenon. The aim of this study is to characterize VIM-52, a His224Arg variant of VIM-1, identified in a Klebsiella pneumoniae clinical isolate. VIM-52 conferred lower MICs to cefepime and ceftazidime as compared to VIM-1. These results were confirmed by steady state kinetic measurements, where VIM-52 yielded a lower activity towards ceftazidime and cefepime but not against carbapenems. Residue 224 is part of the L10 loop (residues 221-241), which borders the active site. As Arg 224 and Ser 228 are both playing an important and interrelated role in enzymatic activity, stability and substrate specificity for the MBLs, targeted mutagenesis at both positions were performed and further confirmed their crucial role for substrate specificity.

Steady-state kinetic analysis of mitochondrial respiratory enzymes from bovine heart mitochondria

Mitochondria is a decisive organelle of cells that produces adenosine triphosphate (ATP) by the process of oxidative phosphorylation of the Krebs cycle and the electron transport chain. The electron transport chain system of mitochondria embodies multiple enzyme supercomplexes including complex I to V which located in the inner membrane. Although the simple enzyme activity of some as-isolated complex has been studied so far, the steady-state kinetic analysis of each complex within the form of mitochondrial supercomplex has not been studied in depth. To this end, kinetic parameters of mitochondrial complex I–IV were determined using steady-kinetic analysis using corresponding substrates of them. Catalytic activity and binding affinity between substrates and enzymes were obtained by fitting the data to the Michaelis–Menten equation. Acquired kinetic parameters represented distinctive values depending on the complexes that can be interpreted by the characteristics of the enzymes including the distinction of substrates or the ratio of the enzyme itself under the supercomplex form. The indirect kcat of the mitochondrial enzymes were varied from 0.0609 to 0.334 s−1 in order of complex III, II, I, and IV and Km of substrates were also diverse from 5.1 μM to 12.14 mM. This is the first attempt to get exact kinetic values that should provide profound information to evaluate the mitochondrial function practically in advance.

L-Cysteine as an Irreversible Inhibitor of the Peroxidase-Mimic Catalytic Activity of 2-Dimensional Ni-Based Nanozymes

The ability to modulate the catalytic activity of inorganic nanozymes is of high interest. In particular, understanding the interactions of inhibitor molecules with nanozymes can bring them one step closer to the natural enzymes and has thus started to attract intense interest. To date, a few reversible inhibitors of the nanozyme activity have been reported. However, there are no reports of irreversible inhibitor molecules that can permanently inhibit the activity of nanozymes. In the current work, we show the ability of L-cysteine to act as an irreversible inhibitor to permanently block the nanozyme activity of 2-dimensional (2D) NiO nanosheets. Determination of the steady state kinetic parameters allowed us to obtain mechanistic insights into the catalytic inhibition process. Further, based on the irreversible catalytic inhibition capability of L-cysteine, we demonstrate a highly specific sensor for the detection of this biologically important molecule.

Exponential Amplification Using Photoredox Autocatalysis

Exponential molecular amplification such as the polymerase chain reaction is a powerful tool that allows ultrasensitive biodetection. Here we report a new exponential amplification strategy based on photoredox autocatalysis, where eosin Y, a photocatalyst, amplifies itself by activating a non-fluorescent eosin Y derivative (EYH2) under green light. The deactivated photocatalyst is stable and rapidly activated under low intensity light, making the eosin Y amplification suitable for resource-limited settings. Through steady-state kinetic studies and reaction modeling, we found that EYH2 is either oxidized to eosin Y via one-electron oxidation by triplet eosin Y and subsequent 1e─/H+ transfer, or activated by singlet oxygen with the risk of degradation. By reducing the rate of the EYH2 degradation, we successfully improved EYH2- to-eosin Y recovery, achieving efficient autocatalytic eosin Y amplification. Additionally, to demonstrate its flexibility in output signals, we coupled the eosin Y amplification with photo-induced chromogenic polymerization, enabling sensitive visual detection of analytes. Finally, we applied the exponential amplification methods in developing bioassays for detection of biomarkers including SARS-CoV-2 nucleocapsid protein, an antigen used in the diagnosis of COVID-19<br>

Exponential Amplification Using Photoredox Autocatalysis

Exponential molecular amplification such as the polymerase chain reaction is a powerful tool that allows ultrasensitive biodetection. Here we report a new exponential amplification strategy based on photoredox autocatalysis, where eosin Y, a photocatalyst, amplifies itself by activating a non-fluorescent eosin Y derivative (EYH2) under green light. The deactivated photocatalyst is stable and rapidly activated under low intensity light, making the eosin Y amplification suitable for resource-limited settings. Through steady-state kinetic studies and reaction modeling, we found that EYH2 is either oxidized to eosin Y via one-electron oxidation by triplet eosin Y and subsequent 1e─/H+ transfer, or activated by singlet oxygen with the risk of degradation. By reducing the rate of the EYH2 degradation, we successfully improved EYH2- to-eosin Y recovery, achieving efficient autocatalytic eosin Y amplification. Additionally, to demonstrate its flexibility in output signals, we coupled the eosin Y amplification with photo-induced chromogenic polymerization, enabling sensitive visual detection of analytes. Finally, we applied the exponential amplification methods in developing bioassays for detection of biomarkers including SARS-CoV-2 nucleocapsid protein, an antigen used in the diagnosis of COVID-19<br>