Enzyme Function Research Articles

Introduction: Cardiac sympathetic innervation plays important roles in regulation of heart rate, contractility, and conduction, as well as postnatal maturation and injury response. However, few physiologically relevant in vitro models of functionally innervated myocardium have been reported to date. Therefore, we sought to engineer a 3D tissue-engineered model of human innervated myocardium to enable functional and pharmacological studies of cardiac innervation. Methods: We first developed a protocol to differentiate functional sympathetic neurons (SNs) from human induced pluripotent stem cells (hiPSCs) using a PHOX2B::eGFP reporter line, followed by characterization by qPCR, immunostaining, and Ca 2+ imaging. Compartmentalized model of innervated engineered cardiac tissues (ECTs) was fabricated using 6-wk old hiPSC-SNs and 3-wk old hiPSC-cardiomyocytes (CMs) transduced with MHCK7-gCaMP6. After 4-6 weeks of culture, structural and functional characterization was performed using immunostaining, force testing, optical mapping, Ca 2+ imaging, and pharmacological tests. Results: hiPSC-SNs expressed canonical transcription factors (Phox2b, Ascl1, Hand2) and functional enzymes (TH, DBH, AChRs), and robustly responded to presynaptic nicotine and electrical stimulation. After 4 weeks of coculture, SN axons were evident in ECT cross-sections (0.23% area) and whole-tissue mounts (4.44% area), indicating successful axon ingrowth. Compared to aneural ECTs, SN-innervated ECTs displayed similar contractile forces and conduction velocities, and 1.32-fold higher Ca 2+ transient amplitudes. Additionally, the spontaneous beating rate and beat-to-beat variability in innervated ECTs was significantly higher than in aneural ECTs (2.1-fold and 2.85-fold, respectively). Upon the addition of 100µM nicotine, the spontaneous beating rate of innervated tissues increased significantly compared to aneural ECTs (1.61-fold), while beat-to-beat variability was unchanged relative to control. Conclusion: Collectively, our results demonstrate the successful generation of an in vitro model of functionally innervated human myocardium, which will enable studies of pathological SN remodeling after myocardial injury and diseases of the heart-brain axis.

Introduction: Diabetic kidney disease (DKD) is one of the most common complication of type 2 diabetes mellitus (T2DM) and confers increased risk of myocardial infarction, stroke, and heart failure. Glucagon-like peptide-1 receptor agonists (GLP-1RAs) and guideline-directed moderate physical training (PT) reduce major adverse cardiovascular events. This study investigated whether this combination provides cardiorenal benefit through improved vascular compliance, renal perfusion, and redox balance. Methods: Male wistar rats were allocated into six groups: controls, GLP-1RA, T2DM, T2DM+PT, T2DM+GLP-1RA and T2DM+PT+GLP-1RA. Interventions lasted 28 days. Renal function (inulin clearance, serum creatinine), microalbuminuria, renal hemodynamics, oxidative biomarkers (urinary peroxides, lipid peroxidation, nitrates, tissue thiols, catalase), and mitochondrial enzyme activity (citrate synthase) were evaluated. In parallel, HK-2 cells were cultured under normoglycemic (5.5 mM) or hyperglycemic (30 mM) conditions and treated with semaglutide (400 nM) for up to 4 days. Cell viability was assessed using MTT assay. Results: The combination of semaglutide and PT in type 2 diabetic rats significantly improved glomerular filtration rate, reduced serum creatinine, microalbuminuria, renal oxidative stress, and vascular resistance, while enhancing renal blood flow and citrate synthase activity. In vitro, semaglutide attenuated high-glucose-induced cytotoxicity in HK-2 cells, preserving cellular viability. Conclusion: Combined treatment with semaglutide and moderate physical training attenuated the progression of DKD induced by T2DM. These findings reinforce the superior renoprotective effects of the combined therapy compared to isolated interventions, particularly through improved renal hemodynamics and reduced oxidative stress. Overall, the data highlights the therapeutic potential of this strategy in mitigating DKD progression and cardiovascular disease.

The β-ketoacyl-acyl carrier protein (ACP) synthases are pivotal elongation enzymes that catalyze the condensation of acyl-CoA or acyl-ACP with malonyl-ACP to produce β-ketoacyl-ACP. Among these, the homologous enzymes FabH (β-ketoacyl-ACP synthase III) and the recently characterized BioZ play crucial roles, initiating the biosynthetic pathways for fatty acids and biotin, respectively. FabH primarily utilizes acetyl-CoA as the primer substrate, whereas BioZ exclusively condenses the longer glutaryl-CoA, which contains a charged ω-carboxyl group. Despite their similar catalytic mechanisms, the molecular bases for the strict substrate specificities remain undetermined. Here, we report crystal structures of the BioZ: glutaryl-CoA cocrystalized complexes and demonstrate the ability to swap the physiological functions and substrate specificities between FabH and BioZ. This functional interchange was achieved by grafting the β8-α9 loop plus residue Ala317 of Agrobacterium tumefaciens BioZ to Escherichia coli FabH, resulting in a shift in substrate preference from acetyl-CoA to glutaryl-CoA. The reverse manipulations of BioZ resulted in FabH activity. These data identify the structural elements as the minimal determinants of substrate specificity and enzyme function. These findings provide valuable insights into the molecular mechanisms of substrate recognition and catalysis by FabH and BioZ and offer a foundation for the development of targeted therapeutic strategies against these enzymes.

Background: Heart failure (HF) with pulmonary hypertension due to left-sided heart disease (PH-LHD) is associated with poor prognosis. Dapagliflozin showed benefits in terms of ejection fraction (EF); meanwhile, sildenafil improved pulmonary pressures and right ventricular function in PH -LHD in recent clinical studies. This study assesses the potential additive effects of dapagliflozin and sildenafil on cardiac function and pulmonary hemodynamics in this population. Methods: In this prospective, randomized, controlled trial, 93 participating patients with HF and PH-LHD were randomly assigned to receive dapagliflozin (control group, n = 48) or dapagliflozin plus sildenafil 25 mg/day (test group, n = 45) in addition to conventional therapy for HF for 12 weeks. The primary outcomes were assessing changes in echocardiographic hemodynamic parameters. Secondary outcomes included outcomes, changes in cardiac enzyme (troponin), kidney function (serum creatinine), and lipid profile. Results: The average baseline median left ventricular ejection fraction (LVEF) for both groups was 30%, and the Pulmonary Artery Systolic Pressure (PASP) median was 50 mmHg. At follow-up, PASP had declined, and EF had improved compared to baseline. However, there were no statistically noticeable variations between the groups (p = 0.458, 0.331, respectively). No notable changes were observed in secondary and safety outcomes, including hospitalization rate, number of deaths, kidney function, and cardiac enzymes (p = 0.524, 1, 0.923, and 0.574, respectively). Conclusions: Addition of sildenafil to dapagliflozin did not demonstrate any significant clinical or hemodynamic benefit over dapagliflozin monotherapy in HF patients and PH-LHD. Further studies are warranted to evaluate the effects over the long term.

This work overviews some complex molecular interactions and phosphorylation events in the insulin receptor (IR) signaling pathway and explains its central role in metabolic control, which starts from the synthesis and secretion of insulin by pancreatic β-cells under elevated blood glucose. The triggered pathway coordinates a cascade of molecular processes that results in the activation of primary metabolic functions. Insulin bound to its receptor starts a sequence of events, such as the autophosphorylation of the receptor β subunit for initiating downstream signaling cascades, glycogen synthesis, and the appropriate regulation of lipid metabolism. The complexity and specificity of the signaling pathway involve insulin receptor substrates, phosphatidylinositol-3-kinase (PI3K), and protein kinase B (PKB). A detailed molecular interaction analysis in the IR has pointed to the crucial role of some residues and structural elements necessary for enzymatic functionalities and substrate binding. These factors include the kinase domain of the IR with specific amino acid residues, and subsequent activation of downstream signaling proteins. The structural changes on phosphorylation promote the binding of SH2 domain-containing adaptor proteins, which lead to the initiation of multifunctional signaling complexes central to insulin signal transduction. These molecular mechanisms provide insight into pathophysiology relating to metabolic diseases and potential treatment targets. This review aims an understanding of insulin receptor operation, elucidating the molecular intricacies behind the eventful metabolic insulin signaling pathways and highlighting possible research and therapeutic development in the field of medicine.

Peptide natural products are important molecules for the development of efficient drugs for human health applications. The biphenomycins are bacterial macrocyclic peptides characterized by unique ortho-tyrosine (oTyr) residues connected by biaryl linkages. Biphenomycins possess potent antibacterial activity against Gram-positive pathogens at low doses with no eukaryotic toxicity. Despite their initial discovery in 1967, their biosynthetic pathway has remained elusive. Within this work, we identified the ribosomal biosynthetic origin of biphenomycins and elucidated all enzymatic maturation steps by in-depth functional characterization in vivo and in vitro. Key steps include selective ortho-hydroxylation events at two phenyl alanine residues catalyzed by a bifunctional multinuclear nonheme iron-dependent oxidase yielding the oTyr functionalities, biaryl cross coupling by a B12-dependent radical SAM enzyme, amino acid side-chain modifications by a highly regioselective arginase and by dedicated hydroxylases, as well as a stepwise proteolytic processing by a TldD-type but self-sufficient protease. These findings clarify the molecular basis of biphenomycin assembly, reveal unprecedented enzymatic dual functions, and provide the foundation for the targeted discovery of novel biphenomycins and for the development of bioengineering strategies to enhance yields and develop antibiotics with further increased potency, addressing the urgent need for new antimicrobial agents.

Optimizing mealworm rearing conditions and gut microbiome function for enhanced plastics biodegradation.

Microplastic pollution in rice systems: Impacts, mechanisms and green remediation strategies.

Global regulator DksA as a key target for naringenin biosynthesis identified via whole-cell directed evolution.

Genome mining of tailoring enzymes from biosynthetic gene clusters for synthetic biology: A case study with fungal methyltransferases.

Small phenolic inhibitors of PfATP6, a Plasmodium falciparum calcium ATPase, as prototype antimalarials.

The effect of cold plasma treatment on the color and drying efficiency of fresh cut yam (Dioscorea opposita Thunb. cv. Tiegun).

Permethrin (PERM) is a synthetic pyrethroid insecticide initially regarded as low risk. However, evidence now indicates that misuse and prolonged exposure can damage multiple physiological systems by disrupting enzymatic functions in subcellular structures. In this study, male Wistar rats were administered PERM (75, 150, or 300 mg/kg/day) for 15 days to assess its effect on hematological and biochemical parameters, including oxidative stress markers in the liver, kidney, and heart. Subacute PERM administration induced significant, dose-dependent toxicological alterations in exposed animals. Hematological analysis revealed impaired hematopoiesis, characterized by increased erythrocytes and platelets alongside decreased hemoglobin, hematocrit, mean corpuscular volume, and red cell distribution width. Biochemical analysis revealed elevated liver enzymes and bilirubin, along with reduced albumin levels, indicating hepatic alterations associated with PERM. The assessment of oxidative stress revealed tissue-specific responses following PERM exposure. While GPx, CAT, and SOD levels remained unchanged, GR activity increased in the heart, and GST activity increased in the liver. Additionally, a substantial decrease in MDA was observed in both the liver and heart. These collective alterations found in PERM-subacute exposed rats suggest the potential for cellular damage with the possible development of chronic pathologies, warranting further investigation.

An adaptive and label-free colorimetric assay for EDTA using copper(II)-aptamer complexes as soft nanozymes.

The crucial necessity to cope with oxidative stress caused not only by external factors but also by the cellular intermediary metabolism has driven the selection of diverse protection mechanisms in strict anaerobic microorganisms. In this context Methanosarcina acetivorans, a marine archaeon, expresses diverse functional enzymes involved in the protection against oxidative stress induced by O2. However, enzymes involved in aldehyde metabolism/detoxification that may have an important role as part of the antioxidant machinery have not yet been evaluated. In this work, the transcriptomic regulation of 8 genes encoding putative aldehyde dehydrogenases (ALDH) and the enzymatic activity were evaluated in M. acetivorans cells grown in the presence of air as an external stressor, and compared with cells grown under anaerobic conditions. The presence of air induced significant higher content of aldehydes, however the air exposition prepared the cells for oxidative stress as judged by changes in the ROS production rate with respect to anaerobic-grown control cells. Transcript levels of 5 ALDH codifying sequences tested were up-regulated, while 3 sequences were down-regulated by the presence of air. Basal ALDH activity levels were detected in anaerobic-grown cells with NAD+ or methylviologen as electron acceptor, suggesting a role of these enzymes in the central metabolism under anaerobic conditions. However, ALDH activity was significantly incremented in cells grown in the presence of air. Incubation of cells with acetaldehyde was toxic for anaerobic cells, but it was tolerated and detoxified in cultures grown with air. Results suggested that in M. acetivorans, ALDHs play a key role metabolizing aldehydes produced as part of the central metabolism, but also in combating oxidative stress induced by air exposure episodes.

Background: Protein arginine deiminase 4 (PAD4) has emerged as a promising therapeutic target for acute promyelocytic leukemia (APL) because of its role in epigenetic regulation and leukemogenesis. All-trans retinoic acid, a standard differentiation agent in APL therapy, has been shown to upregulate PAD4 expression during leukemic cell maturation. Interestingly, first-generation PAD4 inhibitors also promote differentiation, but simultaneously trigger compensatory PAD4 overexpression, underscoring the unresolved complexity of PAD4 modulation in leukemia therapy. Methods: In this study, we employed mass cytometry and transcriptomic–proteomic integrated analysis to investigate the underlying mechanisms of YW3-56, a dual-function PAD4 inhibitor against protein expression and enzymatic function, in NB4 leukemia cells. Functional validation was conducted using Western blot and metabolic assays. Results: Mass cytometry analysis revealed that YW3-56 reduced leukemia stemness (CD44/CD133), while enhancing myeloid differentiation (CD11b/CD14) and immunogenic activation (CD80/CD86). Multiomics analysis revealed a YW3-56-induced metabolic shift characterized by downregulation of glycolytic enzymes and upregulation of the tricarboxylic acid cycle and pentose phosphate pathway components, indicating a reversal of the Warburg effect. Mechanistically, this metabolic reprogramming was driven by reduced AKT expression and phosphorylation at Thr308, impaired GLUT1 expression and membrane localization, and decreased glucose uptake, which collectively promoted the differentiation of NB4 cells. Additionally, YW3-56 suppressed the downstream mTOR pathway, inducing caspase-3/PARP-mediated apoptosis and inhibiting cell proliferation. Conclusions: Our study demonstrated that YW3-56 exerts multimodal antileukemic effects in APL by simultaneously targeting PAD4-mediated epigenetic regulation, AKT-driven metabolic reprogramming and cellular differentiation, highlighting PAD4-AKT signaling as a promising target for APL combination therapy.

Enzyme promiscuity has increasingly garnered significant attention in recent years, with a growing number of enzymes exhibiting this trait being progressively characterized. In plant systems, enzyme promiscuity critically underpins adaptive evolution, where evolutionary pressures drive environmental acclimatization and functional innovation. Conversely, in synthetic biology, this property exhibits a dual-edged nature: broad substrate promiscuity facilitates heterologous biosynthesis of natural products while enabling exploration of novel enzymatic functions and new natural product scaffolds. However, concomitant challenges─including byproduct accumulation, diminished target product purity, and reduced catalytic efficiency stemming from compromised specificity─impede industrial-scale natural product synthesis. Despite these limitations, this inherent enzymatic feature has driven synergistic advancements across biocatalysis, biosynthesis, and protein engineering. This review systematically dissects the manifestations of enzyme promiscuity in microbial natural product biosynthesis and critically evaluates contemporary strategies for alleviating enzyme promiscuity. Ultimately, it seeks to advance mechanistic understanding of enzyme promiscuity and establish a foundational framework for developing high-efficiency biosynthetic platforms.

Reverse triiodothyronine (rT3) is traditionally regarded as a biologically inactive isomer of triiodothyronine (T3), produced primarily via peripheral deiodination of thyroxine (T4). Although T3 binds to nuclear thyroid hormone receptors and regulates transcription of genes essential to neuronal development, synaptic plasticity, and myelination, rT3 lacks such agonist activity. Recent studies suggest rT3 may competitively inhibit T3 at receptor sites and influence thyroid hormone bioavailability by modulating deiodinase enzyme activity. These effects position rT3 as a potentially significant contributor to the pathophysiology of various neuropsychiatric conditions. A comprehensive literature review was conducted through 2024 using biomedical databases including PubMed, Scopus, and Google Scholar. Inclusion criteria targeted peer-reviewed studies investigating rT3 regulation, deiodinase enzyme function (particularly type 3 deiodinase, D3), and clinical correlations between altered thyroid hormone profiles and psychiatric illnesses. Key terms included "reverse T3," "thyroid metabolism," "deiodinase activity," "functional hypothyroidism," and various neuropsychiatric diagnoses. Articles emphasizing both molecular mechanisms and clinical outcomes were prioritized. Increased D3 activity during physiological stress, trauma, chronic inflammation, or psychiatric illness shifts T4 metabolism away from active T3 and toward inactive rT3. This leads to a biochemical state described as "functional hypothyroidism," where serum T4 and thyroid-stimulating hormone (TSH) levels may appear normal, yet intracellular T3 action is diminished. Elevated rT3 and reduced T3/rT3 ratios have been identified in patients with depression, bipolar disorder, generalized anxiety, cognitive impairment, and schizophrenia. These alterations correlate with symptom severity and treatment resistance in some individuals. Additionally, rT3 has shown promise as a biomarker for disrupted thyroid signaling in neuropsychiatric contexts, potentially guiding more personalized treatment approaches. Reverse T3, long viewed as a passive by-product, may play an active regulatory role in neuropsychiatric disorders by interfering with T3 signaling at the cellular level. Functional hypothyroidism driven by excess rT3 represents a distinct biochemical phenotype contributing to mood, cognition, and behavioral dysfunction. Recognition of this mechanism underscores the need for expanded thyroid assessment beyond standard TSH and T4 testing in psychiatric populations. Future research should focus on the therapeutic implications of correcting rT3 dominance and restoring optimal intracellular thyroid hormone activity.

Vitamin B2 (VB2) metabolism regulates numerous cellular processes, but its role in hepatocellular carcinoma (HCC) progression remains unclear. Here we show that HCC tumors are characterized by upregulation of a VB2 metabolism signature, and VB2 metabolism promotes HCC progression. Among VB2 metabolic enzymes, flavin adenine dinucleotide synthase (FADS) is the only one that is widely overexpressed in human HCC. Elevated FADS expression correlates with resistance to anti-PD-1 therapy and poor prognosis. In vivo, FADS facilitates HCC cell growth and suppresses Tcell-mediated antitumor immunity. Single-cell transcriptomic analysis reveals that FADS-induced changes occur both in the tumor cells and the intra-tumoral CD8+ T cells. Knocking down FADS induces HCC cell death and increases CD8⁺ T cell infiltration. Mechanistically, FADS confers ferroptosis resistance on HCC cells via enzymatic function to produce FAD and non-enzymatic function to stabilize PCBP2. Moreover, FADS impairs CD8+ T cell recruitment by disrupting the cGAS-STING pathway. Hesperidin, a clinically approved FADS inhibitor, shows antitumor efficacy in a mouse model. Our study thus highlights the importance of VB2 metabolism in HCC and provides the proof of principle for targeting FADS as a therapeutic strategy for HCC.

In the production of modern nongxiangxing daqu, mechanical stamping is utilized to compact raw materials into daqu bricks. Nevertheless, variations in stamping frequencies may modify the initial physicochemical properties of daqu, which in turn influence its physicochemical and biochemical parameters, and ultimately affect the quality of baijiu. This study systematically evaluated daqu samples prepared with different stamping frequencies (2 to 5 cycles) in terms of (1) physicochemical and biochemical parameters, (2) volatile compound profiles, (3) microbial community dynamics, and (4) interspecific interactions. The results showed that with the increase in stamping frequency, the moisture content, fermentative power, esterifying power, and liquefying power of daqu were all enhanced, with respective increases of 20.11%, 67.16%, 12.24-fold, and 36.27%. Specifically, the relative abundances of Weissella, Lactobacillus, Aspergillus, and Rasamsonia in daqu exhibited a significant increase with the elevation of pressing cycles. With the reduction in stamping frequency, the primary producers of flavor compounds shifted gradually from bacteria to fungi. These findings verify that stamping frequency exert a substantial regulatory impact on the physicochemical and biochemical parameters, microbial community dynamics, accumulation of flavor substances, and abundance of functional enzymes in daqu. Through a systematic elucidation of the mechanistic links between stamping parameters and daqu functionalities, this research offers actionable insights for optimizing industrial pressing processes and establishes a scientific basis for modern daqu production.