Enzyme Substrate Research Articles

The conformational space of RNase P RNA in solution.

RNA conformational diversity has fundamental biological roles1-5, but direct visualization of its full conformational space in solution has not been possible using traditional biophysical techniques. Using solution atomic force microscopy, a deep neural network and statistical analyses, we show that the ribonuclease P (RNase P) RNA adopts heterogeneous conformations consisting of a conformationally invariant core and highly flexible peripheral structural elements that sample a broad conformational space, with amplitudes as large as 20-60 Å in a multitude of directions, with very low net energy cost. Increasing Mg2+ drives compaction and enhances enzymatic activity, probably by narrowing the conformational space. Moreover, analyses of the correlations and anticorrelations between spatial flexibility and sequence conservation suggest that the functional roles of both the structure and dynamics of key regions are embedded in the primary sequence. These findings reveal the structure-dynamics basis for the embodiment of both enzymatic precision and substrate promiscuity in the RNA component of the RNase P. Mapping the conformational space of the RNase P RNA demonstrates a new general approach to studying RNA structure and dynamics.

An Overview of Gaucher Disease

Background: Gaucher disease (GD) is a rare autosomal recessive disorder caused by mutations in the GBA1 gene that lead to a deficiency in the glucocerebrosidase gene. This deficiency results in the accumulation of glucocerebrosides in macrophages, primarily affecting the liver, spleen, and bone marrow. Focusing on the Mexican population, this study aims to review GD’s epidemiology, clinical manifestations, and treatment options to enhance early diagnosis and optimize treatment outcomes. Methods: This study is a comprehensive literature review analyzing epidemiological data, clinical presentations, and current therapeutic approaches for Gaucher disease, including enzyme replacement therapy (ERT) and substrate reduction therapy (SRT). Conclusions: Early diagnosis and individualized treatment, primarily through enzyme replacement therapy, significantly improve the prognosis of patients with Gaucher disease, particularly type 1. Continued research is required to enhance therapeutic approaches for the neuropathic types and better understand the disease’s epidemiology in Mexico. These efforts will contribute to improved clinical outcomes and quality of life for patients.

Broadening the Catalytic Scope of the Peroxygenase Activity of a Bacterial Tyrosine Hydroxylase

AbstractFungal unspecific peroxygenases (UPOs, EC 1.11.2.1) are known for their unique biocatalytic properties and have been extensively studied since their discovery more than two decades ago. UPOs have found a wide range of applications in various fields, such as bioremediation, drug metabolism, and synthetic organic chemistry. Nonetheless, the production of UPOs seems limited to fungal expression systems which makes enzyme engineering challenging. It would be attractive to have a peroxygenase available that is easily expressed in a bacterial host. Here, we report on the expression and engineering of a bacterial peroxygenase, tyrosine hydroxylase (TyrH, EC 1.11.2.6). With only two rounds of computer‐aided engineering, we have identified distal and active‐site mutations that improve enzymatic activities and substrate scope, respectively. The combination of these mutations in a single variant led to an effective peroxygenase, capable of indigo production and enantioselective sulfoxidation and which can be overexpressed in Escherichia coli.

Positive activation entropy of Bacillus circulans xylanase catalyzed ONPX2 hydrolysis: A mechanistic and engineering study

Transition state (TS) stabilization by enzymes greatly accelerates catalytic reactions. For some enzymes, the TS complex has entropy higher than enzyme substrate (ES) complex. But the origin of favorable entropy remains unclear. In this work, we studied the mechanism of Bacillus Circulans xylanase (BCX) 11 catalyzed o-nitrophenyl β-xylobioside (ONPX2) glycoside hydrolysis. The catalytic reaction exhibits a positive activation entropy, and an increase in ionic strength leads to a decrease in entropy without affecting the activation free energy, indicating that the entropy is predominantly influenced by electrostatic forces. Moreover, NMR measurements of electrostatic attractions within the active site demonstrate a positive entropy, aligning with molecular dynamics (MD) simulations showing that electrostatic interactions contribute to the entropic stabilization of the TS complex. These findings suggest that the positive entropy primarily originates from alterations in electrostatic interactions due to the formation of the oxocarbenium ion at C1 in the TS. Differences of electrostatic interactions in ES and TS modify hydrogen bonding of surrounding residues in the active site which causes their side chain dynamics and thus conformational entropy changes. Residues critical for the positive activation entropy are identified. A new BCX mutant with an increased activation entropy and catalytic activity is found.

Another inner truth of shaking: Water migration and transformation-advanced physicochemical alterations in tea leaves

Shaking, essential in oolong tea production, is becoming an innovative method to impart floral fragrance. Research on shaking primarily concentrates on biological underpinnings, including modifications in gene expression and stress-triggered enzymatic catalysis, and consequent physicochemical properties. Water phase and distribution, reshaped by shaking and affected the biological and physicochemical alterations of tea leaves, is always ignored. This work utilized TEM, LF-NMR, UPLC-QqQ-MS, and GC-TOF-MS to explore physicochemical alterations during shaking. Results revealed shaking induced stomatal opening, water migration from stems to leaf veins, and a reduction in free water, transformed into bound water. Mechanical stimulation disrupted cell microstructures, including vacuoles, chloroplasts, and cell walls, releasing precursors and enzyme substrates. Shaking triggered intracellular physicochemical reactions that decreased polyphenols, amino acids, chlorophyll, and carotenoids, while increasing organic acids and sugars. Also catalyzed the synthesis of aromatic compounds like (E)-nerolidol, β-ionone epoxide, and α-farnesene, shaping the floral-fruity aroma and mellow taste of tea.

Gas Empowered Dual‐Cascade Strategy for Augmented Single‐Atom Nanotherapies

AbstractSingle‐atom nanotherapies have received numerous attention in malignant oncotherapy. However, the insufficient enzyme substrate and the upregulation of heat shock proteins during therapeutic interventions are seldom concurrently noticed. Herein, a novel gas empowered dual‐cascade synergistic treatment strategy is demonstrated with domino effect, which can sequentially reinforce single‐atom nanozyme (SAzyme)‐based enzymatic therapeutics and mild photothermal therapy (PTT) (< 45 °C). In the proof‐of‐concept study, Fe single atom nanozyme (Fe/SAzyme) loaded with hydrogen sulfide (H2S) donor NaHS is developed for HSPs‐silencing mediated mild PTT. The generated H2S suppresses the catalase activity to achieve “intracellular H2O2 conservation”, thereby furnishing the enzyme substrate to Fe/SAzyme to produce abundant cytotoxic hydroxyl radicals (·OH) for augmented enzymatic therapeutics. Then, excess ·OH induced mitochondrial dysfunction blocks adenosine triphosphate (ATP) energy supply to realize cellular energy remodeling, which hinders overexpression of HSPs and enhances mild PTT of Fe/SAzyme both in vitro and vivo. Consequently, the gas‐triggered dual‐cascade strategy achieves domino H2S/·OH/mitochondrial dysfunction synergistic effect, endowing SAzymes with maximum antitumor efficacy via enzymatic therapeutics combined with mild PTT. This dual‐cascaded gas/enzymatic/mild PTT synergistic oncotherapy not only exhibits a new pathway for gas‐facilitated mild PTT, but also offers a valuable paradigm for the application of “1 + 1 + 1 > 3” multimodal synergistic tumor therapy.

Enhanced chitinase production by Bacillus paralicheniformis GXMU-J23.1: Optimization, genomic insights, and chitin degradation mechanism

Millions of tons of shrimp and crab waste, rich in chitin, are produced annually worldwide. To efficiently utilize this resource and address the contamination caused by traditional chitin treatment, a high-chitinase-producing strain, GXMU-J23.1, was isolated from the marine environment and identified as Bacillus paralicheniformis. Genome sequencing revealed several chitinolytic enzymes, such as chitinase, chitin deacetylase, and polysaccharide monooxygenases. Under optimal conditions, the chitinase activity increased 9.1-fold to 356.32 ± 1.21 U/mL. The purified chitinase Chi23 exhibited optimal activity at 50 °C and pH 5.0, degrading various chitin substrates. Metal ions such as Ca2+ and reagents such as EDTA increased the activity, whereas Fe2+ and Zn2+ inhibited the activity. Chi23, an endochitinase, converts chitin into chitotriose and diacetylchitobiose. Based on the structural reconstruction and molecular docking of Chi23, the potential enzyme–substrate mode of action was elucidated, which will support subsequent enzyme modification and in-depth development of enzyme systems assisting in chitin degradation.

Metabolic fingerprinting reveals roles of Arabidopsis thaliana BGLU1, BGLU3, and BGLU4 in glycosylation of various flavonoids

Flavonoids are specialized metabolites that play important roles in plants, including interactions with the environment. The high structural diversity of this metabolite group is largely due to enzyme-mediated modifications of flavonoid core skeletons. In particular, glycosylation with different sugars is very common. In this study, the functions of the Arabidopsis thaliana glycoside hydrolase family 1-type glycosyltransferase proteins BGLU1, BGLU3, and BGLU4 were investigated, using a reverse genetics approach and untargeted metabolic fingerprinting. We screened for metabolic differences between A. thaliana wild type, loss-of-function mutants, and overexpression lines and partially identified differentially accumulating metabolites, which are putative products and/or substrates of the BGLU enzymes. Our study revealed that the investigated BGLU proteins are glycosyltransferases involved in the glycosylation of already glycosylated flavonoids using different substrates. While BGLU1 appears to be involved in the rhamnosylation of a kaempferol diglycoside in leaves, BGLU3 and BGLU4 are likely involved in the glycosylation of quercetin diglycosides in A. thaliana seeds. In addition, we present evidence that BGLU3 is a multifunctional enzyme that catalyzes other metabolic reactions with more complex substrates. This study deepens our understanding of the metabolic pathways and enzymes that contribute to the high structural diversity of flavonoids.

Structure-based clustering and mutagenesis of bacterial tannases reveals the importance and diversity of active site-capping domains.

Tannins are critical plant defense metabolites, enriched in bark and leaves, that protect against microorganisms and insects by binding to and precipitating proteins. Hydrolyzable tannins contain ester bonds which can be cleaved by tannases-serine hydrolases containing so-called "cap" domains covering their active sites. However, comprehensive insights into the biochemical properties and structural diversity of tannases are limited, especially regarding their cap domains. We here present a code pipeline for structure prediction-based hierarchical clustering to categorize the whole family of bacterial tannases, and have used it to discover new types of cap domains and other structural insertions among these enzymes. Subsequently, we used two recently identified tannases from the gut/soil bacterium Clostridium butyricum as model systems to explore the biochemical and structural properties of the cap domains of tannases. We demonstrate using molecular dynamics and mutagenesis that the cap domain covering the active site plays a major role in enzyme substrate preference, inhibition, and activity-despite not directly interacting with smaller substrates. The present work provides deeper knowledge into the mechanism, structural dynamics, and diversity of tannases. The structure-based clustering approach presents a new way of classifying any other enzyme family, and will be of relevance for enzyme types where activity is influenced by variable loop or insert regions appended to a core protein fold.

The Trajectory of Damaged-Base Eversion into the Active Site of Apurinic/Apyrimidinic Endonuclease APE1 Regulates This Enzyme's Substrate Specificity.

Apurinic/apyrimidinic endonuclease 1 (APE1) is responsible for the hydrolysis of the phosphodiester bond on the 5' side of an apurinic/apyrimidinic site during base excision repair. Moreover, in DNA, this enzyme can recognize nucleotides containing such damaged bases as 5,6-dihydro-2'-deoxyuridine (DHU), 2'-deoxyuridine (dU), alpha-2'-deoxyadenosine (αA), and 1,N6-ethenoadenosine (εA). Previously, by pulsed electron-electron double resonance spectroscopy and pre-steady-state kinetic analysis, we have revealed multistep DNA rearrangements during the formation of the catalytic complex. In the present study, the modeling of the eversion trajectory of nucleotides with various damaged bases was performed by directed molecular dynamics simulations. It was found that each damaged base at the beginning of the eversion interacts with protein loop Val196-Arg201, which should be moved to enable further nucleotide eversion. This movement involves a shift in loop Val196-Arg201 away from loop Asn253-Thr257 and requires the disruption of contacts between these loops. The Glu260Ala substitution facilitates the separation of the two loops. Moreover, conformational changes in the Asn253-Thr257 loop should occur in the second half of the lesion eversion trajectory. All these perturbations within the protein globule tend to reduce steric interactions of each damaged base with the protein during the eversion of the nucleotide from DNA and movement to the active site. These perturbations are important determinants of substrate specificity of endonuclease APE1.

Recent Therapeutic Advancements for Gaucher Disease

AbstractGaucher Disease (GD) is a well‐known lysosomal storage disease resulting from mutations in the GBA1 gene. GD exhibits a range of clinical manifestations, each with unique symptoms and severity levels. This review explores the genetic foundations of GD, highlighting the significance of Glucocerebrosidase (GCase) deficiency, resulting in the skeletal complications associated with GD, such as osteonecrosis, fractures, and bone pain, all of which significantly negatively influence the quality of life for patients. Over 700 mutations in the GBA1 gene are found to cause variations in gene expression in GD, indicating the disease's complexity and the need for continued research. Early diagnosis and prognosis evaluation depend heavily on diagnostic approaches integrating laboratory assessments, genetic testing, and clinical symptoms. Treatment strategies like enzyme replacement therapy (ERT) and substrate reduction therapy (SRT) have advanced, but issues like high costs and invasiveness still exist. This review focuses on novel therapeutic approaches that show promise in treating GD, including gene and cell‐based therapies, pharmacological chaperone therapy (PCT), and drug delivery via nanoparticles. Finally, discussions on current clinical trials, limitations of the advanced therapies, and future scope are summarized.

Charge‐Mediated Interactions Affect Enzymatic Reactions in Peptide Condensates

AbstractBiomolecular condensates, formed through liquid‐liquid phase separation (LLPS), serve as enzymatic reaction centers in cells by increasing local concentrations of enzymes and substrates, thereby facilitating reaction kinetics and regulatory mechanisms. Inspired by these natural systems, synthetic condensates are being developed for diverse applications, including payload delivery, sensing, and as microreactors where enzymatic reaction kinetics can be modulated by factors like pH, viscosity, and enzyme‐substrate co‐localization. Here, we investigate how the physicochemical properties of enzymes and substrates influence condensate formation and function as microreactors. Focusing on cellulase and alkaline phosphatase, which differ in molecular weight and isoelectric point, we employed a minimalistic complex coacervation system of oppositely charged LLPS‐promoting peptides. Our findings show how electrostatic forces within condensates influence their role as microreactors. Specifically, the ability of condensates to encapsulate or exclude phosphatase, cellulase, and their substrates, which is pivotal for the regulation of reaction kinetics, is determined by the enzyme surface charge, substrate charge, and condensate charge stoichiometry. These results highlight the potential of utilizing electrostatic forces within condensates to modulate enzymatic reactions, providing critical insights for developing synthetic condensates as microreactors in biotechnology and materials science.

Impact of Flooding on Lands with Emerging Contaminants on the Quality of Receiving Water Bodies

Among crucial factors that control flooding events are extreme climate, urban growth, and mismanagement. Islands in the Nile River have experienced successive cycles of flooding and drying due to recent rainy years in the Nile Basin countries. This paper focuses on the impact of floods on the amounts of ammonium-N released, total concentrations of heavy metals, enzyme activities, and microbial biomass (C and N) in flood soils of 11 Nile River islands. Field moist soils and their airdried counterparts were collected from the outskirts of the island and incubated for 15 days at 30 °C to be analyzed. Results reflected that the amounts of released NH4-N were higher in airdried than in wet soils. The average hydrolysis rates of the studied six enzyme substrates were correlated significantly with organic C, organic N, microbial bio-mass C and N in the field moist soils. The highest correlation coefficients (r) were with rates of hydrolysis of substrates of asparaginase, amidase, and β-glycosaminidase. A significant intercorrelation between rates of hydrolysis of the six studied enzyme substrates and r values which range from 0.81 to 0.98. At 10 mmol kg−1 soil, Co+2, Cd+2, Pb+2, Cu+2, Cr+3, and Ni+2 inhibited the release of NH4-N. Arginine hydrolysis was inhibited by the six heavy metals at 5 mmol kg−1 soil; the most effective inhibitors were Pb+2, Ni+2, and Cr+3. Due to the complex soil enzymatic and organic nitrogen hydrolase activities that occur during floods, fertile fields on the Nile River islands should not be fertilized before the summer flood seasons, which will prevent contamination of the Nile River water and loss of its island soil fertility.

Prediction of polybrominated diphenyl ethers (PBDEs) as potential substrates of various human CYP enzymes and laboratory test of BDE-99 for its metabolism-activated mutagenicity

Polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants, of which BDE-47 could be activated by human cytochrome P450s (CYPs) for chromosome-damaging effects. However, the metabolic activation and mutagenicity of other PBDEs remain unknown. In this study, 14 representative PBDEs were analyzed by molecular docking as potential substrates for several human CYPs. The results showed negative free energies for each pair of binding, however, different CYPs demonstrated largely varied frequencies of binding conformations favoring a substrate potential: CYP2E1, 3A4, and 2B6 being suitable for all/most compounds. Using BDE-99 (5 ∼ 40 μM) as a model compound (exposing for 2 cell cycles), it did not induce micronucleus in a human hepatoma HepG2 cell line, however, positive result was observed in C3A cells (derived from HepG2 but with enhanced expression of CYPs). Pretreatment of HepG2 cells with each of bisphenol A (1 μM, inducer of CYPs) and CITCO (10 μM, inducer of CYP2B6) led to micronucleus formation by BDE-99, while the effect of BDE-99 in C3A cells was abolished by 1-aminobenzotriazole (60 μM, inhibitor of CYPs). In a V79-derived cell line genetically engineered for expressing human CYP2B6 BDE-99 induced micronucleus, while it was negative in V79-Mz and its derivatives expressing several other human CYPs. The micronuclei formed in HepG2 cells pretreated with BPA and CITCO were free of centromere protein B immunofluorescence staining. Finally, BDE-99 weakly induced PIG-A gene mutations in C3A, while negative in HepG2 cells. In conclusion, our study suggest that BDE-99 may be activated by human CYP2B6 for chromosome-breaking effects.

Mutations Selectively Evolving Peroxidase Activity Among Alternative Catalytic Functions of Human Glutathione Transferase P1-1.

Glutathione transferases are detoxication enzymes with broad catalytic diversity, and small alterations to the protein's primary structure can have considerable effects on the enzyme's substrate selectivity profile. We demonstrate that two point mutations in glutathione transferase P1-1 suffice to generate 20-fold enhanced non-selenium-dependent peroxidase activity indicating a facile evolutionary trajectory. Designed mutant libraries of the enzyme were screened for catalytic activities with alternative substrates representing four divergent chemistries. The chemical reactions comprised aromatic substitution, Michael addition, thiocarbamoylation, and hydroperoxide reduction. Two mutants, R1 (Y109H) and an R1-based mutant V2 (Q40M-E41Q-A46S-Y109H-V200L), were discovered with 16.3- and 30-foldincreased peroxidase activity with cumene hydroperoxide (CuOOH) compared to the wildtype enzyme, respectively. The basis of the improved peroxidase activity of the mutant V2 was elucidated by constructing double-point mutants. The mutants V501 (Q40M-Y109H) and V503 (E41Q-Y109H) were found to have 20- and 21-fold improvements in peroxidase activity relative to the wildtype enzyme, respectively. The steady-state kinetic profiles of mutants R1 and V2 in the reduction of CuOOH were compared to the wildtype parameters. The kcat values for R1 and V2 were 34- and 57-fold higher, respectively, than that of the wildtype enzyme, whereas the mutant Km values were increased approximately 3-fold. A 10-fold increased catalytic efficiency (kcat/Km) in CuOOH reduction is accomplished by the Tyr109His point mutation in R1. The 23-fold increase of the efficiency obtained in V2 was caused by adding further mutations primarily enhancing kcat. In all mutants with elevated peroxidase activity, His109 played a pivotal role.

Crystal Structure and Mutagenesis of an XYP Subfamily Cyclodipeptide Synthase Reveal Key Determinants of Enzyme Activity and Substrate Specificity.

Cyclodipeptide synthases (CDPSs) catalyze the synthesis of diverse cyclodipeptides (CDPs) by utilizing two aminoacyl-tRNA (aa-tRNA) substrates in a sequential ping-pong reaction mechanism. Numerous CDPSs have been characterized to provide precursors for diketopiperazines (DKPs) with diverse structural characteristics and biological activities. BcmA, belonging to the XYP subfamily, is a cyclo(l-Ile-l-Leu)-synthesizing CDPS involved in the biosynthesis of the antibiotic bicyclomycin. The structural basis and determinants influencing BcmA enzyme activity and substrate selectivity are not well understood. Here, we report the crystal structure of SsBcmA from Streptomyces sapporonensis. Through structural comparison and systematic site-directed mutagenesis, we highlight the significance of key residues located in the aminoacyl-binding pocket for enzyme activity and substrate specificity. In particular, the nonconserved residues D161 and K165 in pocket P2 are essential for the activity of SsBcmA without significant alteration of the substrate specificity, while the conserved residues F158 as well as F210 and S211 in P2 are responsible for determining substrate selectivity. These findings facilitate the understanding of how CDPSs selectively accept hydrophobic substrates and provide additional clues for the engineering of these enzymes for synthetic biology applications.

Exploring Gaucher disease: A pediatric case of severe dyslipidemia and unique symptoms

Introduction: Gaucher disease is a rare inherited lysosomal storage disorder caused by mutations in the GBA gene, leading to glucocerebrosidase deficiency. This results in the accumulation of glucocerebroside in macrophages, forming "Gaucher cells" that infiltrate various tissues, primarily affecting the liver, spleen, bone marrow, and nervous system. The disease manifests in three types, with Type 1 being the most common and devoid of neurological symptoms. Diagnosis involves enzyme level testing, genetic screening, and imaging, while treatment primarily consists of enzyme replacement therapy (ERT) and substrate reduction therapy (SRT). Case-Report: A one-year-old girl presented with weakness, irritability, and recurrent fever over six months, previously misdiagnosed with malaria and respiratory infections. Physical examination revealed pallor, hepatosplenomegaly, and cherry-red spots on fundoscopy. Laboratory findings indicated anemia, leukocytosis, and severe dyslipidemia, highlighting atypical features for Gaucher disease. Discussion: This case emphasizes the diagnostic challenges in pediatric Gaucher disease, especially with nonspecific symptoms. The combination of cherry-red spots and dyslipidemia suggests a unique variant or severity of the disease, underlining the necessity for heightened awareness among healthcare providers for prompt diagnosis. Conclusion: Gaucher disease should be considered in pediatric patients with unexplained systemic symptoms. Early detection and ERT initiation can significantly improve clinical outcomes. The case illustrates the variability in clinical presentations, emphasizing the need for comprehensive diagnostic evaluation.

Protein Quakes in Redox Metalloenzymes: Clues to Molecular Enzyme Conductivity Triggered by Binding of Small Substrate Molecules.

Multicentre redox metalloproteins undergo conformational changes on electrochemical surfaces, or on enzyme substrate binding. The two-centre copper enzymes, laccase (Type I and TypeII/III Cu) and nitrite reductase (CuNIR) (Type I and Type II Cu) are examples. With some exceptions, these enzymes show no non-turnover voltammetry on Au(111)-surfaces modified by thiol based self-assembled molecular monolayers, but dioxygen or nitrite substrate triggers strong electrocatalytic signals. Scanning tunnelling microscopy also shows high conductivity only when dioxygen or nitrite is present. Atomic force microscopy shows constant CuNIR height but pronounced structural expansion in the electrocatalytic range on nitrite binding. We have recently offered a rationale, based on ab initio quantum chemical studies of water/nitrite substitution in a 740-atom CuNIR fragment. Presently we provide much more detailed structural assignment mapped to single-residue resolution. NO2 --binding induces both a 2 Å Cu-Cu distance increase, and pronounced frontier orbital delocalization strongly facilitating ET between the Cu regions. The conformational changes transmit from the catalytic Type II centre to the electron inlet Type I centre, via the His129-Cys130 ligands, and via Type I-Cys130 or Type I-His129 ending at Type II Asp92. The ET patterns are reflected in different atomic Mulliken charges in the water and nitrite CuNIR fragment.

CIGB-300 internalizes and impairs viability of NSCLC cells lacking actionable targets by inhibiting casein kinase-2 signaling

Overall response rates in advanced Non-Small Cell Lung Cancer (NSCLC) remains low. Thus, novel molecular targets, tailored drugs and/or drug combinations are needed. Casein Kinase-2 (CK2) is a constitutively active and frequently over-expressed enzyme which fosters tumor survival, proliferation and metastasis. By using a clinical-grade and Cell Penetrating Peptide-based inhibitor coined as CIGB-300, we explore the anti-neoplastic effects caused by interruption of CK2 signaling in lung cancer cells lacking EGFR, ALK and ROS mutations. CIGB-300 penetrated and impaired viability and proliferation of Lung Adenocarcinoma (LUAD) (A549, NCI-H522) and Lung Squamous Carcinoma (LUSC) (NCI-H226 and SK-MES-1) cells in a dose-response manner. The differential activity could not be explained by overall peptide uptake or its subcellular distribution, as evidenced by flow cytometry and confocal microscopy. Upon internalization, CIGB-300 interacted with CK2 catalytic subunits (ɑ1/ɑ2) and CK2 substrates, thus impairing phosphorylation of enzyme substrates (CDC37s13, NPM1s125) and downstream proteins (RPS6s325/326). CK2 inhibition induced an early Reactive Oxygen Species (ROS) and mitochondrial membrane depolarization, which predates lung cancer cell death. Finally, intravenous injection of CIGB-300 in a cell line-based xenograft corroborated CIGB-300’s anti-tumor effects and suggested concurrent in situ reductions of CSNK2ɑ subunit and downstream RPS6s235/236 phosphorylation. Overall, CIGB-300 therapeutic hypothesis and antineoplastic effects demonstrated herein, further support the evaluation of this clinical-grade CK2 inhibitor in advanced NSCLC with limited therapeutic options.

A New Fluorescent Method for Measuring Peroxiredoxin Enzyme Activity Using Monobromobimane.

A novel fluorometric method is presented for accurately quantifying peroxiredoxin (Prx) enzyme activity in vitro. The rate-limiting step in the Prx-catalyzed reaction is the dissociation of peroxide. To avoid interference from catalase, we developed an assay using tert-butyl hydroperoxide (t-BOOH) as a substrate for specific Prx activity measurement. The assay involves incubating the enzyme substrates 1,4-dithio-DL-threitol (DTT) and t-BOOH in a suitable buffer at 37°C for 10min in a known volume of Prx enzyme. Following incubation, the reagent monobromobimane (mBB) is added to terminate the enzymatic reaction and produce a fluorescent product. Prx activity is subsequently determined by measuring thiol fluorescence, with reaction conditions optimized using a Bland-Altman plot. The efficacy of this novel protocol was rigorously validated by comparing Prx activity measurements from paired samples with those generated by a reference assay. A correlation coefficient of 0.995 was observed between the two methods, demonstrating superior precision and reliability compared to existing methods. The mBB-Prx protocol offers a significant safety advantage by using t-BOOH as a substrate for Prx activity measurement. As catalase does not catalyze t-BOOH dissociation, including sodium azide is unnecessary. Moreover, the method obviates the need for concentrated acids to terminate the Prx enzymatic reaction, as the mBB reagent efficiently inhibits Prx activity. This streamlined approach simplifies the assay and significantly improves its safety and usability, providing users with a reliable and convenient tool. The convenience of this method allows users to focus on their research without worrying about safety or complex procedures.