Phosphorylation Of Substrates Research Articles

Growth response of Bacillus coagulans on TiO2 nanoparticles by RNA-Seq analysis of the customary probiotic strain

The phosphoenolpyruvate sugar phosphotransferase systems in bacteria are complex enzyme systems carrying out various important functions like detection, phosphorylation and transport of several sugar substrates including mono and disaccharides, amino sugars, etc. This key enzyme complex prevents the efflux of sugar substrates by phosphorylation and prepares them for energy production and metabolism. In this study, a high throughput RNA sequencing method was used to compare the gene expression in Bacillus coagulans treated with nano titanium dioxide (TiO2) rutile. Out of 3175 genes, 499 were differentially expressed. Gene Ontology analysis revealed 239 significant genes. In the biological process category, we found the phosphoenolpyruvate-dependent sugar phosphotransferase systems are the dominant ones and are involved in the uptake of specific carbohydrate sources. In our previous study, we showed increased growth of bacteria in presence of nano TiO2 anatase with a sharp rise in ATP concentrations. We know that, among metal oxide-based nanoparticles, TiO2 is placed at the topmost position in the agri-food sector based on its use and their reactivity with human beneficial gut flora, commonly known as probiotics, becoming a major concern. Hence, the interaction of TiO2 rutile nanoparticles on Bacillus coagulans was studied thoroughly at the transcriptomics level; with depth.

A General Light‐Driven Organocatalytic Platform for the Activation of Inert Substrates

AbstractDue to their strong covalent bonds and low reduction potentials, activating inert substrates is challenging. Recent advances in photoredox catalysis offered a number of solutions, each of which useful for activating specific inert bonds. Developing a general catalytic platform that can consistently target a broad range of inert substrates would be synthetically useful. Herein, we report a readily available indole thiolate organocatalyst that, upon excitation with 405 nm light, acquires a strongly reducing power. This excited‐state reactivity served to activate, by single‐electron reduction, strong C−F, C−Cl, and C−O bonds in both aromatic and aliphatic substrates. This catalytic platform was versatile enough to promote the reduction of generally recalcitrant electron‐rich substrates (Ered<−3.0 V vs SCE), including arenes that afforded 1,4‐cyclohexadienes. The protocol was also useful for the borylation and phosphorylation of inert substrates with a high functional group tolerance. Mechanistic studies identified an excited‐state thiolate anion as responsible of the highly reducing reactivity.

A General Light-Driven Organocatalytic Platform for the Activation of Inert Substrates.

Due to their strong covalent bonds and low reduction potentials, activating inert substrates is challenging. Recent advances in photoredox catalysis offered a number of solutions, each of which useful for activating specific inert bonds. Developing a general catalytic platform that can consistently target a broad range of inert substrates would be synthetically useful. Herein, we report a readily available indole thiolate organocatalyst that, upon excitation with 405 nm light, acquires a strongly reducing power. This excited-state reactivity served to activate, by single-electron reduction, strong C-F, C-Cl, and C-O bonds in both aromatic and aliphatic substrates. This catalytic platform was versatile enough to promote the reduction of generally recalcitrant electron-rich substrates (Ered <-3.0 V vs SCE), including arenes that afforded 1,4-cyclohexadienes. The protocol was also useful for the borylation and phosphorylation of inert substrates with a high functional group tolerance. Mechanistic studies identified an excited-state thiolate anion as responsible of the highly reducing reactivity.

1593-P: Insulin-Degrading Enzyme (IDE) Regulates Glucagon Signalling and Mitochondrial Respiration in Hepatocytes

IDE is a ubiquitous metalloprotease with cytosolic and mitochondrial subcellular localization that degrades insulin and glucagon. People with T2D and diet-induced obese mice show lower hepatic IDE levels. We revealed a key role of IDE on the insulin-mediated repression of hepatic gluconeogenesis, but its function on glucagon-dependent activation of gluconeogenesis and mitochondrial respiration in hepatocytes remains completely unknown. Here, we aim to elucidate the role of IDE on glucagon signalling and its impact on gluconeogenesis and energy metabolism in hepatocytes. Liver homogenized and primary hepatocytes obtained from L-IDE-KO mice (deletion of IDE in liver) showed decreased expression of glucagon receptor (~60%), CREB protein (~40%), and lower phosphorylation of CREB (~50%) upon glucagon stimulation compared to controls. Similar results were found in AML12-shRNA-Ide cells, in which IDE protein levels were reduced by ~50%. Additionally, glucagon stimulation resulted in lower (~30%) cAMP levels and diminished phosphorylation of PKA substrates in AML12-shRNA-Ide. Surprisingly, these alterations in glucagon signalling paralleled with ~20-fold increases in the expression of the gluconeogenic genes G6p6 and Pck1. Of note, basal and uncoupler-stimulated respiration increased ~4-fold in AML12-shRNA-Ide in parallel with a ~2-fold increment of mitochondrial and glycolytic ATP production. Finally, similar mitochondrial phenotype was found in human hepatocytes lacking IDE (HepG2-IDE-KO cells), which exhibited higher FoxO1 levels and fragmented mitochondria. The effects on mitochondrial respiration were independent of IDE's proteolytic activity. In summary, reduced IDE expression in hepatocytes has a deleterious effect on glucagon signalling leading to up-regulated gluconeogenesis and mitochondrial respiration. We conclude that IDE is a mechanistic link to couple hepatic gluconeogenesis with mitochondrial energy production. Disclosure C.M. González-Casimiro: None. P. Cámara-Torres: None. B. Merino: None. J. Santo-Domingo: None. M.A. de la Fuente: None. A. Alonso: None. I. Cozar-Castellano: None. G. Perdomo: None. Funding Ministerio de Ciencia e Innovación-Spain (PID2019-110496RB-C22)

Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein.

The activation of at least 23 different mammalian kinases requires the phosphorylation of their hydrophobic motifs by the kinase PDK1. A linker connects the phosphoinositide-binding PH domain to the catalytic domain, which contains a docking site for substrates called the PIF pocket. Here, we used a chemical biology approach to show that PDK1 existed in equilibrium between at least three distinct conformations with differing substrate specificities. The inositol polyphosphate derivative HYG8 bound to the PH domain and disrupted PDK1 dimerization by stabilizing a monomeric conformation in which the PH domain associated with the catalytic domain and the PIF pocket was accessible. In the absence of lipids, HYG8 potently inhibited the phosphorylation of Akt (also termed PKB) but did not affect the intrinsic activity of PDK1 or the phosphorylation of SGK, which requires docking to the PIF pocket. In contrast, the small-molecule valsartan bound to the PIF pocket and stabilized a second distinct monomeric conformation. Our study reveals dynamic conformations of full-length PDK1 in which the location of the linker and the PH domain relative to the catalytic domain determines the selective phosphorylation of PDK1 substrates. The study further suggests new approaches for the design of drugs to selectively modulate signaling downstream of PDK1.

The GCN2/eIF2αK stress kinase regulates PP1 to ensure mitotic fidelity.

GCN2/eIF2αK4 is exclusively seen as an eIF2α kinase, which regulates reprogramming of protein translation in response to stress. Here, we show that GCN2 has an unexpected role in unstressed cells as a regulator of mitosis. This function is not through its canonical role in translation reprogramming, but through the regulation of two previously unidentified substrates, PP1α and γ. In the absence of GCN2 function, timing and levels of phosphorylation of key mitotic players are altered, leading to aberrant chromosome alignment, missegregating chromosomes, elevated number of tripolar spindles, and a delay in progression through mitosis. Pharmacological inhibition of GCN2 results in similar effects and is synergistic with Aurora A inhibition in causing more severe mitotic errors and cell death. We suggest that GCN2-dependent phosphorylation of PP1α and γ restrains their activity and this is important to ensure the timely regulation of phosphorylation of several PP1 substrates during early mitosis. These findings highlight a druggable PP1 inhibitor and open new avenues of research on the therapeutic potential of GCN2 inhibitors.

Rapid adaptation to CDK2 inhibition exposes intrinsic cell-cycle plasticity

CDK2 is a core cell-cycle kinase that phosphorylates many substrates to drive progression through the cell cycle. CDK2 is hyperactivated in multiple cancers and is therefore an attractive therapeutic target. Here, we use several CDK2 inhibitors in clinical development to interrogate CDK2 substrate phosphorylation, cell-cycle progression, and drug adaptation in preclinical models. Whereas CDK1 is known to compensate for loss of CDK2 in Cdk2-/- mice, this is not true of acute inhibition of CDK2. Upon CDK2 inhibition, cells exhibit a rapid loss of substrate phosphorylation that rebounds within several hours. CDK4/6 activity backstops inhibition of CDK2 and sustains the proliferative program by maintaining Rb1 hyperphosphorylation, active E2F transcription, and cyclin A2 expression, enabling re-activation of CDK2 in the presence of drug. Our results augment our understanding of CDK plasticity and indicate that co-inhibition of CDK2 and CDK4/6 may be required to suppress adaptation to CDK2 inhibitors currently under clinical assessment.

Bisphenol S Reduces Pig Spermatozoa Motility through Different Intracellular Pathways and Mechanisms than Its Analog Bisphenol A.

Bisphenol A (BPA: 2,3-bis (4-hydroxyphenyl) propane) is an environmental chemical widely used in the manufacturing of epoxy polymers and many thermoplastic consumer products. Serious concerns about its safety led to the development of analogs, such as BPS (4-hydroxyphenyl sulfone). Very limited studies about BPS's impact on reproduction, specifically in spermatozoa, exist in comparison with BPA. Therefore, this work aims to study the in vitro impact of BPS in pig spermatozoa in comparison with BPA, focusing on sperm motility, intracellular signaling pathways and functional sperm parameters. We have used porcine spermatozoa as an optimal and validated in vitro cell model to investigate sperm toxicity. Pig spermatozoa were exposed to 1 and 100 μM BPS or BPA for 3 and 20 h. Both bisphenol S and A (100 μM) significantly reduce pig sperm motility in a time-dependent manner, although BPS exerts a lower and slower effect than BPA. Moreover, BPS (100 μM, 20 h) causes a significant increase in the mitochondrial reactive species, whereas it does not affect sperm viability, mitochondrial membrane potential, cell reactive oxygen species, GSK3α/β phosphorylation or phosphorylation of PKA substrates. However, BPA (100 μM, 20 h) leads to a decrease in sperm viability, mitochondrial membrane potential, GSK3β phosphorylation and PKA phosphorylation, also causing an increase in cell reactive oxygen species and mitochondrial reactive species. These intracellular effects and signaling pathways inhibited might contribute to explaining the BPA-triggered reduction in pig sperm motility. However, the intracellular pathways and mechanisms triggered by BPS are different, and the BPS-caused reduction in motility can be only partially attributed to an increase in mitochondrial oxidant species.

Inactivation of Minar2 in mice hyperactivates mTOR signaling and results in obesity

ObjectiveObesity is a complex disorder and is linked to chronic diseases such as type 2 diabetes. Major intrinsically disordered NOTCH2-associated receptor2 (MINAR2) is an understudied protein with an unknown role in obesity and metabolism. The purpose of this study was to determine the impact of Minar2 on adipose tissues and obesity. MethodWe generated Minar2 knockout (KO) mice and used various molecular, proteomic, biochemical, histopathology, and cell culture studies to determine the pathophysiological role of Minar2 in adipocytes. ResultsWe demonstrated that the inactivation of Minar2 results in increased body fat with hypertrophic adipocytes. Minar2 KO mice on a high-fat diet develop obesity and impaired glucose tolerance and metabolism. Mechanistically, Minar2 interacts with Raptor, a specific and essential component of mammalian TOR complex 1 (mTORC1) and inhibits mTOR activation. mTOR is hyperactivated in the adipocytes deficient for Minar2 and over-expression of Minar2 in HEK-293 cells inhibited mTOR activation and phosphorylation of mTORC1 substrates, including S6 kinase, and 4E-BP1. ConclusionOur findings identified Minar2 as a novel physiological negative regulator of mTORC1 with a key role in obesity and metabolic disorders. Impaired expression or activation of MINAR2 could lead to obesity and obesity-associated diseases.

The NMDA receptor regulates integrin activation, ATP release and arterial thrombosis through store-operated Ca2+ entry in platelets.

Platelet activation and thrombus formation is crucial for hemostasis, but also trigger arterial thrombosis. Calcium mobilization plays an important role in platelet activation, because many cellular processes depend on the level of intracellular Ca2+ ([Ca2+](i)), such as integrin activation, degranulation, cytoskeletal reorganization. Different modulators of Ca2+ signaling have been implied, such as STIM1, Orai1, CyPA, SGK1, etc. Also, the N-methyl-D-aspartate receptor (NMDAR) was identified to contribute to Ca2+ signaling in platelets. However, the role of the NMDAR in thrombus formation is not well defined. In vitro and in vivo analysis of platelet-specific NMDAR knock-out mice. In this study, we analyzed Grin1fl/fl-Pf4-Cre+ mice with a platelet-specific knock-out of the essential GluN1 subunit of the NMDAR. We found reduced store-operated Ca2+ entry (SOCE), but unaltered store release in GluN1-deficient platelets. Defective SOCE resulted in reduced Src and PKC substrate phosphorylation following stimulation of glycoprotein (GP)VI or the thrombin receptor PAR4 followed by decreased integrin activation but unaltered degranulation. Consequently, thrombus formation on collagen under flow conditions was reduced ex vivo, and Grin1fl/fl-Pf4-Cre+ mice were protected against arterial thrombosis. Results from human platelets treated with the NMDAR antagonist MK-801 revealed a crucial role of the NMDAR in integrin activation and Ca2+ homeostasis in human platelets as well. NMDAR signaling is important for SOCE in platelets and contributes to platelet activation and arterial thrombosis. Thus, the NMDAR represents a novel target for anti-platelet therapy in cardiovascular disease (CVD).

Sudachitin and Nobiletin Stimulate Lipolysis via Activation of the cAMP/PKA/HSL Pathway in 3T3-L1 Adipocytes.

Polymethoxyflavones are flavonoids that are abundant in citrus fruit peels and have beneficial effects on human health. Previous studies have demonstrated that the polymethoxyflavones, namely sudachitin and nobiletin, ameliorate obesity and diabetes in humans and rodents. Although nobiletin induces lipolysis in adipocytes, lipolytic pathway activation by sudachitin has not been clarified in adipocytes. In this study, the effect of sudachitin on lipolysis was elucidated in murine 3T3-L1 adipocytes. Glycerol release into the medium and activation of the cyclic AMP (cAMP)/protein kinase A (PKA)/hormone-sensitive lipase (HSL) pathway was evaluated in 3T3-L1-differentiated adipocytes. Treatment with sudachitin and nobiletin for 24 and 48 h did not induce cytotoxicity at concentrations of up to 50 μM. Sudachitin and nobiletin at concentrations of 30 and 50 μM increased intracellular cAMP and medium glycerol levels in 3T3-L1 adipocytes. Western blotting revealed that sudachitin and nobiletin dose-dependently increased protein levels of phosphorylated PKA substrates and phosphorylated HSL. Sudachitin- and nobiletin-induced glycerol release, phosphorylation of PKA substrates, and HSL phosphorylation were suppressed by pharmacological inhibition of adenylate cyclase and PKA. These findings indicated that sudachitin, similar to nobiletin, exerts anti-obesogenic effects, at least in part through the induction of lipolysis in adipocytes.

The Role of Polo-Like Kinase 1 in Regulating the Forkhead Box Family Transcription Factors.

Polo-like kinase 1 (PLK1) is a serine/threonine kinase with more than 600 phosphorylation substrates through which it regulates many biological processes, including mitosis, apoptosis, metabolism, RNA processing, vesicle transport, and G2 DNA-damage checkpoint recovery, among others. Among the many PLK1 targets are members of the FOX family of transcription factors (FOX TFs), including FOXM1, FOXO1, FOXO3, and FOXK1. FOXM1 and FOXK1 have critical oncogenic roles in cancer through their antagonism of apoptotic signals and their promotion of cell proliferation, metastasis, angiogenesis, and therapeutic resistance. In contrast, FOXO1 and FOXO3 have been identified to have broad functions in maintaining cellular homeostasis. In this review, we discuss PLK1-mediated regulation of FOX TFs, highlighting the effects of PLK1 on the activity and stability of these proteins. In addition, we review the prognostic and clinical significance of these proteins in human cancers and, more importantly, the different approaches that have been used to disrupt PLK1 and FOX TF-mediated signaling networks. Furthermore, we discuss the therapeutic potential of targeting PLK1-regulated FOX TFs in human cancers.

The ribosomal S6 kinase 2 (RSK2)–SPRED2 complex regulates the phosphorylation of RSK substrates and MAPK signaling

Sprouty-related EVH-1 domain-containing (SPRED) proteins are a family of proteins that negatively regulate the RAS-Mitogen-Activated Protein Kinase (MAPK) pathway, which is involved in the regulation of the mitogenic response and cell proliferation. However, the mechanism by which these proteins affect RAS-MAPK signaling has not been elucidated. Patients with mutations in SPRED give rise to unique disease phenotypes; thus, we hypothesized that distinct interactions across SPRED proteins may account for alternative nodes of regulation. To characterize the SPRED interactome and evaluate how members of the SPRED family function through unique binding partners, we performed affinity purification mass spectrometry. We identified 90-kDa ribosomal S6 kinase 2 (RSK2) as a specific interactor of SPRED2 but not SPRED1 or SPRED3. We identified that the N-terminal kinase domain of RSK2 mediates the interaction between amino acids 123 to 201 of SPRED2. Using X-ray crystallography, we determined the structure of the SPRED2-RSK2 complex and identified the SPRED2 motif, F145A, as critical for interaction. We found that the formation of this interaction is regulated by MAPK signaling events. We also find that this interaction between SPRED2 and RSK2 has functional consequences, whereby the knockdown of SPRED2 resulted in increased phosphorylation of RSK substrates, YB1 and CREB. Furthermore, SPRED2 knockdown hindered phospho-RSK membrane and nuclear subcellular localization. We report that disruption of the SPRED2-RSK complex has effects on RAS-MAPK signaling dynamics. Our analysis reveals that members of the SPRED family have unique protein binding partners and describes the molecular and functional determinants of SPRED2-RSK2 complex dynamics.

STK33 Phosphorylates Fibrous Sheath Protein AKAP3/4 to Regulate Sperm Flagella Assembly in Spermiogenesis

Spermatogenesis defects are important for male infertility; however, the etiology and pathogenesis are still unknown. Herein, we identified two loss-of-function mutations of STK33 in seven individuals with non-obstructive azoospermia. Further functional studies of these frameshift and nonsense mutations revealed that Stk33-/KI male mice were sterile, and Stk33-/KI sperm were abnormal with defects in the mitochondrial sheath, fibrous sheath, outer dense fiber, and axoneme. Stk33KI/KI male mice were subfertile and had oligoasthenozoospermia. Differential phosphoproteomic analysis and invitro kinase assay identified novel phosphorylation substrates of STK33, fibrous sheath components A-kinase anchoring protein 3 and A-kinase anchoring protein 4, whose expression levels decreased in testis after deletion of Stk33. STK33 regulated the phosphorylation of A-kinase anchoring protein 3/4, affected the assembly of fibrous sheath in the sperm, and played an essential role in spermiogenesis and male infertility.

Liver acts as a metabolic gate for the traumatic brain injury pathology: Protective action of thyroid hormone

Clinical evidence indicates that injury to the brain elicits systemic metabolic disturbances that contributes to the brain pathology. Since dietary fructose is metabolized in the liver, we explored mechanisms by which traumatic brain injury (TBI) and dietary fructose influence liver function and their possible repercussions to brain. Consumption of fructose contributed to the detrimental effects of TBI on liver operation, in terms of glucose and lipid metabolism, de novo lipogenesis, lipid peroxidation. Thyroid hormone (T4) is metabolized in the liver and found that T4 supply improved lipid metabolism by reducing de novo lipogenesis, lipid accumulation, lipogenic enzymes (ACC, AceCS1, FAS), lipid peroxidation in liver in response to fructose and fructose-TBI. T4 supply also helped to normalize glucose metabolism and improve insulin sensitivity. Furthermore, T4 counteracted elevations of the pro-inflammatory cytokines, Tnfα and Mcp-1 after TBI and/or fructose intake in liver and circulation. T4 also exerted an effect on isolated primary hepatocytes by potentiating phosphorylation of AMPKα and AKT substrate, AS160, leading to increased glucose uptake. In addition, T4 restored the metabolism of DHA in the liver disrupted by TBI and fructose, adding important information to optimize the action of DHA in therapeutics. The overall evidence seems to indicate that the liver works as a gate for the regulation of the effects of brain injury and foods on brain pathologies.

Abstract 123: Protein Kinase C Theta (PKCθ) Signaling Associated With The Generation Of Procoagulant Platelets Revealed By Phosphoproteomics And Systems Analysis

Protein kinase C (PKC) family members integrate platelet activation programs to support hemostasis, and, also drive inflammatory and vascular diseases. While some PKC isoforms have established roles in platelets, roles for PKCθ are less specified. Here, we aim to determine how PKCθ signaling orchestrates platelet function. We prepared washed platelets from human donors for biochemical, physiological and omics studies. Platelet signaling and physiology were studied following stimulation with agonists, including the glycoprotein receptor GPVI agonist crosslinked collagen-related peptide (CRP-XL), and protease activated receptor PAR1 and PAR4 agonists, TRAP6 and AYPGKF-NH 2 . Platelets were treated with inhibitors of PKC family members to study PKC activation mechanisms by Western blot using antisera against specific phosphorylation sites on PKC isoforms and PKC substrates. Phosphoproteomics studies of GPVI and PAR agonist-stimulated platelets quantified significant, site-specific changes in the phosphorylation of PKC isoforms as well as PKC substrates. In addition to established phosphorylation events (e.g., PKCδ Tyr311), we noted a novel, prominent phosphorylation on the C-terminus PKCθ at Ser685. After generating and characterizing antisera specifically reactive to PKCθ phospho-Ser685, Western blot analysis determined that PKCθ Ser685 phosphorylation occurred later following GPVI platelet activation (&gt;5 min) relative to PKCδ Tyr311 phosphorylation and PKC substrate phosphorylation driven by calcium-dependent PKCs. Treatment of platelets with specific PKC inhibitors revealed that PKCα/β, PKCθ, and, to a lesser extent, PKCδ activities all determine PKCθ Ser685 phosphorylation. In parallel, platelet function studies found that inhibition of PKCθ had only minor effects on platelet secretion and aggregation; however, PKCθ inhibition significantly reduced platelet phosphatidylserine exposure - a marker of procoagulant platelet activity - in response to GPVI and PAR agonists. Together, our results suggest that PKCδ and PKCα/β activities drive the phosphorylation of PKCθ Ser685 to support procoagulant platelet generation in a manner that may be blunted by therapeutically relevant small molecule kinase inhibitors.

Dissecting the effects of GTPase and kinase domain mutations on LRRK2 endosomal localization and activity

Parkinson's disease-causing leucine-rich repeat kinase 2 (LRRK2) mutations lead to varying degrees of Rab GTPase hyperphosphorylation. Puzzlingly, LRRK2 GTPase-inactivating mutations-which do not affect intrinsic kinase activity-lead to higher levels of cellular Rab phosphorylation than kinase-activating mutations. Here, we investigate whether mutation-dependent differences in LRRK2 cellular localization could explain this discrepancy. We discover that blocking endosomal maturation leads to the rapid formation of mutant LRRK2+ endosomes on which LRRK2 phosphorylates substrate Rabs. LRRK2+ endosomes are maintained through positive feedback, which mutually reinforces membrane localization of LRRK2 and phosphorylated Rab substrates. Furthermore, across a panel of mutants, cells expressing GTPase-inactivating mutants form strikingly more LRRK2+ endosomes than cells expressing kinase-activating mutants, resulting in higher total cellular levels of phosphorylated Rabs. Our study suggests that the increased probability that LRRK2 GTPase-inactivating mutants are retained on intracellular membranes compared to kinase-activating mutants leads to higher substrate phosphorylation.

The toxicity of dysregulated Plk1 activity revealed by its suppressor mutations.

Polo-like kinase 1 (Plk1) is a mitotic kinase that has multiple functions throughout the cell cycle. Catalytic activation of Plk1 is known to be regulated by phosphorylation of the kinase domain, including Thr210, and by releasing the kinase domain from its inhibitory polo-box domain. However, how Plk1 is activated to fulfill its proper roles, in time and space, is not well understood. In this study, we unintentionally found that the expression of a constitutively active form of human Plk1 is toxic to bacterial cells, such that cells contained point mutations that alleviate the kinase activity. Structural prediction revealed that these mutations are adjacent to the amino acids supporting the kinase activity. When human cells express these mutants, we found decreased levels of Plk1's substrate phosphorylation, resulting in mitotic defects. Moreover, unlike in bacterial cells, the expression of activated Plk1 mutants did not affect cell proliferation in human cells unless localized at the right place in mitosis. Our observations identified new suppressor mutations and underscored the importance of spatiotemporal regulation in Plk1, providing a basis for how we might intervene in this kinase for therapeutic purpose in human cells.

Zr4+-mediated DNAzyme-driven DNA walker amplification strategy for electrochemical assay of protein kinase a activity and inhibition

Protein kinase A (PKA) can regulate many cellular biological processes by phosphorylation substrate peptide or protein. Sensitive detection of PKA activity is critical for the PKA-related drug discovery and disease diagnosis. A new electrochemical biosensing method was developed for detection of PKA activity based on Zr4+-mediated DNAzyme-driven DNA walker signal amplification strategy. In this strategy, the special designed substrate peptide and a thiolated methylene blue-labeled hairpin DNA (MB-hpDNA) containing a single ribonucleic acid group (rA) could be anchored on the surface of gold electrode by Au–S bond. In the presence of adenosine triphosphate (ATP) and PKA, substrate peptide was phosphorylated and linked with walker DNA (WD) via the robust phosphate-Zr4+-phosphate chemistry. The linked WD hybridized with the loop region of MB-hpDNA to form a Mn2+-dependent deoxynuclease (DNAzyme), which cleaved the MB-hpDNA into MB-labeled fragment releasing away from electrode surface, resulting in a dramatic decrease of electrochemical signal and providing an electrochemical sensing platform for PKA activity detection. The response signal of the developed biosensor is proportional to the logarithm of PKA concentration in the range of 0.05 U mL−1 to 100 U mL−1, with a detection limit of 0.017 U mL−1 at a signal to noise ratio of 3. Furthermore, the proposed method can also be applied for the evaluation of PKA inhibition and PKA activity assay in cell samples. Therefore, the proposed biosensor shows great promise as a universal tool for diagnostics and drug discovery of PKA-related diseases.

Irreversible biosynthesis of D-Allulose from D-glucose in Escherichia coli through fine-tuning of carbon flux and cofactor regeneration engineering.

As a rare hexose with low calories and various physiological functions, D-allulose has drawn increasing attention. The current industrial production of D-allulose from D-fructose or D-glucose is achieved via epimerization based on the Izumoring strategy; however, the inherent reaction equilibrium during reversible reaction limits its high conversion yield. Although the conversion of D-fructose to D-allulose could be enhanced via phosphorylation-dephosphorylation mediated by metabolic engineering, biomass reduction and byproduct accumulation remain a largely unresolved issue. After modifying the glycolytic pathway of Escherichia coli and optimizing the whole-cell reaction condition, the engineered strain produced 7.57 ± 0.61 g L-1 D-allulose from 30 g L-1 D-glucose after 24 h of catalysis. By developing an ATP regeneration system for enhanced substrate phosphorylation, the cell growth inhibition was alleviated and D-allulose production increased by 55.3% to 11.76 ± 0.58 g L-1 (0.53 g g-1 ). Fine-tuning of carbon flux caused a 48% reduction in D-fructose accumulation to 1.47 ± 0.15 g L-1 . After implementing fed-batch co-substrate strategy, the D-allulose titer reached 15.80 ± 0.31 g L-1 (0.62 g g-1 ) with a D-glucose conversion rate of 84.8%. This study presented a novel strategy for high-yield D-allulose production from low-cost substrate. This article is protected by copyright. All rights reserved.