Enzymatic Dephosphorylation Research Articles

Effect of protein charge manipulation on αs1-casein-enriched protein self-assembly

Enzymatic dephosphorylation of casein removes phosphate groups from serine residues and reduces the negative charge. In contrast, succinylation caps the ε-amino group of lysine residues and increases the negative charge on the caseins. The effect of these modifications on the self-association of αs1-casein was studied using analytical ultracentrifugation. Dephosphorylation and succinylation have contrasting effects on αs1-casein self-assembly. Native αs1-casein was a mixture of dimers, trimers and higher order oligomers under these experimental conditions. Increasing the level of succinylation dissociated oligomeric αs1-casein resulting in an increase in the monomeric protein. In contrast, dephosphorylated samples formed larger assemblies compared to the native αs1-casein. Experiments performed at protein concentrations of 1, 2 or 3 mg mL−1 provided consistent results indicating that across this concentration range there is no major difference in the assembly formation. This study demonstrated the utility of analytical ultracentrifugation to understand casein assembly, which will underpin the understanding of proteins structures in dairy foods.

Assessment of the Enzymatic Dephosphorylation Kinetics in the Assemblies of a Phosphopentapeptide that Forms Intranuclear Nanoribbons.

Although the formation of peptide assemblies catalyzed by alkaline phosphatase (ALP) has received increasing attention in inhibiting cancer cells, the detailed enzyme kinetics of the dephosphorylation of the corresponding phosphopeptide assemblies have yet to be determined. We recently discovered that assemblies from a phosphopentapeptide can form intracellular nanoribbons that kill induced pluripotent stem cells or osteosarcoma cells, but the kinetics of enzymatic dephosphorylation remain unknown. Thus, we chose to examine the enzyme kinetics of the dephosphorylation of the phosphopentapeptide [NBD-LLLLpY (1)] from concentrations below to above its critical micelle concentration (CMC). Our results show that the phosphopeptide exhibits a CMC of 75 μM in phosphate saline buffer, and the apparent Vmax and Km values of alkaline phosphatase catalyzed dephosphorylation are approximately 0.24 μM/s and 5.67 mM, respectively. Despite dephosphorylation remaining incomplete at 60 min in all the concentrations tested, dephosphorylation of the phosphopeptide at concentrations of 200 μM or above mainly results in nanoribbons, dephosphorylation at concentrations of CMC largely produces nanofibers, and dephosphorylation below the CMC largely generates nanoparticles. Moreover, the formation of nanoribbons correlates with the intranuclear accumulation of the pentapeptide. By providing the first examination of the enzymatic kinetics of phosphopeptide assemblies, this work further supports the notion that the assemblies of phosphopentapeptides can act as a new functional entity for controlling cell fates.

Enzymatic self-assembly of short peptides for cell spheroid formation.

Cell spheroids, including organoids, serve as a valuable link between in vitro systems and in vivo animal models, offering powerful tools for studying cell biology in a three-dimensional environment. However, existing methods for generating cell spheroids are time consuming or difficult to scale up for large-scale production. Our recent study has revealed that transcytotic peptide assemblies, which transform from nanoparticles to nanofibers by enzymatic reactions, can create an intercellular fibril/gel, accelerating cell spheroid formation from a 2D cell culture or a cell suspension. While this finding presents an alternative approach for generating cell spheroids, the specific structural features required for efficient cell spheroid formation remain unclear. Based on our observation that a phosphotetrapeptide with a biphenyl cap at its N-terminus enables cell spheroid formation, we produced 10 variants of the original peptide. The variants explored modifications to the peptide backbone, length, electronic properties of the biphenyl capping group, and the type of phosphorylated amino acid residue. We then evaluated their ability for inducing cell spheroid formation. Our analysis revealed that, among the tested molecules, peptides with C-terminal phosphotyrosine, low critical micelle concentration, and dephosphorylation-guided nanoparticle to nanofiber morphological transition were the most effective in inducing the formation of cell spheroids. This work represents the first example to correlate the thermodynamic properties (e.g., self-assembling ability) and kinetic behavior (e.g., enzymatic dephosphorylation) of peptides with the efficacy of controlling intercellular interaction, thus offering valuable insights into using enzymatic self-assembly to generate peptide assemblies for biological applications.

Effect of enzymatic dephosphorylation on caprine casein micelle structure

The effect of enzymatic dephosphorylation, using intestinal alkaline phosphatase, on the structure of casein micelles in caprine micellar casein concentrate (MCC) was studied. An optimal condition, involving preheating the MCC dispersion, pH 6.4, 2.5 mg casein/mL and 0.4 U phosphatase/mL, was established and used to prepare MCC with 0–49% dephosphorylation by incubation at 37 °C for 0–180 min. β-Casein showed marked dephosphorylation and formed multi-phosphorylated isoforms depending on dephosphorylation degree, whereas αs- and κ-caseins showed limited and fast dephosphorylation, respectively. With increasing dephosphorylation, both the colloidal calcium and the calcium sensitive micellar caseins, especially β-casein, were gradually dissociated, and the calcium insensitive serum κ-casein was gradually associated with the micelles. The dissociated β-casein fraction was predominated by isoforms with lower number of phosphate groups. For micelles with increasing dephosphorylation, the molar mass decreased, the gyration and hydrodynamic radii decreased, the ratio of gyration to hydrodynamic radii and micellar hydration increased, the spherical morphology was generally retained, and the internal protein inhomogeneity disappeared progressively. These results suggest that after dephosphorylation, the caprine micelle framework underwent an intra-micellar mass redistribution, and become more loose and homogeneous.

Nucleoside Triphosphate Diphosphohydrolase 1 Exhibits Enzymatic Activity toward Tenofovir Diphosphate.

Tenofovir (TFV; prescribed as TFV disoproxil fumarate and TFV alafenamide prodrugs) is currently used for HIV prevention and treatment. TFV must be phosphorylated twice into TFV-diphosphate (TFV-DP) to become pharmacologically active. Previously, we reported heterogeneity in TFV-DP distribution in colorectal tissue (a putative site of HIV infection) sections collected from research participants receiving a TFV-containing enema. This observed heterogeneity is likely multifactorial. Of note, TFV-DP is structurally similar to ATP. It is known that nucleotidases such as nucleoside triphosphate diphosphohydrolases (NTPDases) dephosphorylate ATP. Thus, it was hypothesized that NTPDase-mediated dephosphorylation plays a role in regulating TFV-DP levels in colorectal tissue. To test this hypothesis, recombinant NTPDase proteins (NTPDase 1, 3, 4, 5, 6, and 8) were incubated, individually, with TFV-DP to determine their abilities to dephosphorylate TFV-DP in vitro. Following incubations, TFV-DP dephosphorylation was determined using both malachite green phosphate assays and ultrahigh-performance liquid chromatography tandem mass spectrometry. From these, NTPDase 1 exhibited the highest activity toward TFV-DP. Further, enzyme kinetic analysis revealed Michaelis-Menten kinetics for NTPDase 1-mediated TFV-DP dephosphorylation. Next, immunoblot analyses were conducted to confirm the expression of NTPDase 1 protein in human colorectal tissue. Liquid chromatography coupled to mass spectrometry proteomics analysis was used to measure the relative abundance of NTPDases in human colorectal tissue among healthy adult individuals (n = 4). These analyses confirmed the high abundance of NTPDase 1 in human colorectal tissue. Taken together, results suggest that NTPDase 1 may contribute to the regulation of TFV-DP levels. The above data provide important insights into the dephosphorylation of TFV-DP. SIGNIFICANCE STATEMENT: Nucleoside triphosphate diphosphohydrolases (NTPDases) that are involved in enzymatic ATP dephosphorylation may contribute to tenofovir-diphosphate (TFV-DP) dephosphorylation, leading to its inactivation. In this study, the NTPDases responsible for TFV-DP dephosphorylation in vitro and their expression in human colorectal tissue were investigated. Through this work, it was demonstrated that NTPDase 1 has the highest activity toward TFV-DP dephosphorylation, and it was abundant in human colorectal tissue. Importantly, these studies will increase our understanding of TFV-DP disposition.

Abstract 3454: Adenosine receptor antagonists A2AR (TT-10) and A2BR (TT-4) demonstrate anti-tumor activity in 4T1-induced syngeneic breast cancer mouse model

Abstract Background: Extracellular adenosine has been shown to negatively regulate anti-tumor immunity. Adenosine is produced at high levels in the TME as a result of hypoxia-driven tumor cell death. As a result, ATP is released from dying cells generating adenosine via enzymatic dephosphorylation, mainly by ectonucleotidases CD39 & CD73. This results in high extracellular adenosine levels, being elevated 100-500x in the TME. The free adenosine proceeds to binding one of its four known receptors A1, A2a, A2b & A3. The binding of adenosine to A2AR & A2BR contributes to immunosuppression via cyclic AMP pathway. It has been observed that bound A2AR suppress T cell function (CD8+T & NK) while A2BR dampens myeloid cell function and promotes chemotaxis. These concerted actions thereby enhance tumor growth and metastasis. Blocking the binding of adenosine to the A2AR & A2BR results in T-cell activation and infiltration of immune cells in the TME. TT-10 & TT-4 are potent and selective antagonists of A2AR & A2BR. Both agents are being developed for the treatment of advanced cancers individually and as dual inhibitors. Methods: Balb/c mice were implanted with 5 x 104 4T1 cells and randomly assigned to 6 groups per study; (1) Vehicle Control-VC, (2) TT-10, 1 mg/kg, PO, BID (3) TT-4, 3 mg/kg, PO, BID (4) Anti-mPD-1, 200 ug, IP (D4, 8, 14) (5) 1 mg/kg TT-10 + 200 ug Anti-mPD-1 (D4, 8, 14) and (6) TT-4, 3 mg/kg, PO, BID + 200 ug Anti-mPD-1 (D4, 8, 14). Mice were dosed for 21 days, and tumor volume was measured 2x a week. After 21 days post initiation of treatment (31 days post 4T1 cell inoculation), mice were sacrificed, TIL populations were isolated from 4T1 induced tumors and interrogated via Flow. Markers for surface staining included CD45, CD3, CD4, CD8, CD11b, CD49, Ly6G. Results: All implanted mice developed measurable tumors. Mean suppression of tumor growth was observed to be greater in single agent TT-10 (p<0.05) & TT-4 (p<0.05), over single agent anti-PD-1 inhibitor when compared to the control on day 31, post 4T1 cell inoculation. Analysis of the TIL at the end of the study further supported the observation of tumor growth suppression, from increased T-cell activity seen for both TT-10 & TT-4 when compared to the VC. Furthermore, striking reductions in MDSC populations were also observed in both TT-10 & TT-4 treated mice, when compared to single agent anti-PD-1 and the VC. Conclusions: TT-10 & TT-4 alone was superior (p<0.05) to the VC as well as single agent anti-PD-1 in slowing tumor growth, which was further supported by the increase in T-cell activity observed in the end of study TIL analysis. Lastly, striking reductions in MDSC populations were also observed in both TT-10 & TT-4 treated mice, when compared to single agent anti-PD-1 and the VC. Citation Format: Desa Rae E. Pastore, Kasim Mookhtiar, Brian Schwartz, Sushant Kumar, Ranganayaki Nagaraj, Ashwinkumar V. Meru. Adenosine receptor antagonists A2AR (TT-10) and A2BR (TT-4) demonstrate anti-tumor activity in 4T1-induced syngeneic breast cancer mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3454.

Synthesis and bioactivity of pyrrole-conjugated phosphopeptides.

Here we report the synthesis and effect on the cell viability of pyrrole-conjugated phosphopeptides. Encouraged by the selective inhibition of cancer cells by a naphthyl-capped phosphopeptide (Nap-ffpy, 1), we conjugated the heteroaromatic dipyrrole or tripyrrole motif at the N-terminal of short peptides containing phosphotyrosine or phosphoserine and examined the bioactivity of the resulting phosphopeptides (2–10). Although most of the phosphopeptides exhibit comparable activities with that of 1 against HeLa cells at 200 μM, they, differing from 1, are largely compatible with HeLa cells at 400 μM. Enzymatic dephosphorylation of 2–10, at 400 μM is unable to induce a dramatic morphological transition of the peptide assemblies observed in the case of 1. These results suggest that a heteroaromatic motif at the N-terminal of peptides likely disfavors the formation of extensive nanofibers or morphological changes during enzymatic self-assembly, thus provide useful insights for the development of phosphopeptides as substrates of phosphatases for controlling cell fate.

Bioorthogonal Disassembly of Tetrazine Bearing Supramolecular Assemblies Inside Living Cells.

Supramolecular assemblies are an emerging class of nanomaterials for drug delivery systems (DDS), while their unintended retention in the biological milieu remains largely unsolved. To realize the prompt clearance of supramolecular assemblies, the bioorthogonal reaction to disassemble and clear the supramolecular assemblies within living cells is investigated here. A series of tetrazine-capped assembly precursors which can self-assemble into nanofibers and hydrogels upon enzymatic dephosphorylation are designed. Such an enzyme-instructed supramolecular assembly process can perform intracellularly. The time-dependent accumulation of assemblies elicits oxidative stress and induces cellular toxicity. Tetrazine-bearing assemblies react with trans-cyclooctene derivatives, which lead to the disruption of π-π stacking and induce disassembly. In this way, the intracellular self-assemblies disassemble and are deprived of potency. This bioorthogonal disassembly strategy leverages the biosafety aspect in developing nanomaterials for DDSs.

In vitro assessment of enzymatic phytate dephosphorylation during digestive process of different feeds and feed ingredients

In vivo studies of the digestive process are long, expensive and difficult to rationalize, whereas in vitro systems may give more accessible insight into parts of this process. The purpose of this study was to show the ability of a three-step simulation of monogastric animals’ digestive system to estimate phytate hydrolysis and how it is affected by feed composition. Several feed ingredients: wheat, maize, soybean meal and rapeseed meal and complete diets: a wheat-maize-soybean-meal-based diet, a maize-soybean-meal diet and a wheat-maize-rapeseed-meal diet were treated using an adaptation of a described in vitro digestion simulation system in the presence of increasing doses of phytase. A strong dependence of phytate hydrolysis on the feed ingredient used was obtained: phosphorus releases were 0.3, 0.8, 1.0 and 1.6 g/kg at 0 U/kg of phytase supplementation for maize, soybean meal, wheat and rapeseed meal respectively and 1.2, 2.9, 1.7 and 3.9 at 1000 U/kg of bacterial phytase. The efficacy of enzymatic dephosphorylation of phytate was found dependent on the ingredient, which can be partially explained by their initial content in myo-inositol phosphates. The in vitro simulation was proven a useful tool to assess enzymatic dephosphorylation of phytate under different conditions.

Dephosphorylation of pyridoxal 5′‐phosphate‐derived Schiff bases in the presence of bovine alkaline phosphatase

AbstractThe present paper reports on the study of the dephosphorylation of pyridoxal 5′‐phosphate and four derived hydrazones (containing the residues of pyrazine, 2‐furan, 2‐thiophene, 3‐pyridine carboxylic acids) induced by bovine alkaline phosphatase from intestinal mucosa at 298.2 K and pH 10 (0.05 m Tris–HCl buffer). We observed and discussed characteristic changes in the UV–vis and fluorescent spectra of substrates. Michaelis–Menten parameters of the enzymatic dephosphorylation are calculated. The stability of alkaline phosphatase in the presence of hydrazones is confirmed. The dephosphorylation of the Zn(II) complex with pyridoxal 5′‐phosphate‐derived hydrazone is analyzed.

Enzymatically Forming Intranuclear Peptide Assemblies for Selectively Killing Human Induced Pluripotent Stem Cells.

Tumorigenic risk of undifferentiated human induced pluripotent stem cells (iPSCs), being a major obstacle for clinical application of iPSCs, requires novel approaches for selectively eliminating undifferentiated iPSCs. Here, we show that an l-phosphopentapeptide, upon the dephosphorylation catalyzed by alkaline phosphatase (ALP) overexpressed by iPSCs, rapidly forms intranuclear peptide assemblies made of α-helices to selectively kill iPSCs. The phosphopentapeptide, consisting of four l-leucine residues and a C-terminal l-phosphotyrosine, self-assembles to form micelles/nanoparticles, which transform into peptide nanofibers/nanoribbons after enzymatic dephosphorylation removes the phosphate group from the l-phosphotyrosine. The concentration of ALP and incubation time dictates the morphology of the peptide assemblies. Circular dichroism and FTIR indicate that the l-pentapeptide in the assemblies contains a mixture of an α-helix and aggregated strands. Incubating the l-phosphopentapeptide with human iPSCs results in rapid killing of the iPSCs (=<2 h) due to the significant accumulation of the peptide assemblies in the nuclei of iPSCs. The phosphopentapeptide is innocuous to normal cells (e.g., HEK293 and hematopoietic progenitor cell (HPC)) because normal cells hardly overexpress ALP. Inhibiting ALP, mutating the l-phosphotyrosine from the C-terminal to the middle of the phosphopentapeptides, or replacing l-leucine to d-leucine in the phosphopentapeptide abolishes the intranuclear assemblies of the pentapeptides. Treating the l-phosphopentapeptide with cell lysate of normal cells (e.g., HS-5) confirms the proteolysis of the l-pentapeptide. This work, as the first case of intranuclear assemblies of peptides, not only illustrates the application of enzymatic noncovalent synthesis for selectively targeting nuclei of cells but also may lead to a new way to eliminate other pathological cells that express a high level of certain enzymes.

Partial-, Double-Enzymatic Dephosphorylation and EndoGluC Hydrolysis as an Original Approach to Enhancing Identification of Casein Phosphopeptides (CPPs) by Mass Spectrometry.

The identification of phosphopeptides is currently a challenge when they are part of a complex matrix of peptides, such as a milk protein enzymatic hydrolysate. This challenge increases with both the number of phosphorylation sites on the phosphopeptides and their amino acid length. Here, this paper reports a four-phase strategy from an enzymatic casein hydrolysate before a mass spectrometry analysis in order to enhance the identification of phosphopeptides and phosphosites: (i) the control protein hydrolysate, (ii) a two-step enzymatic dephosphorylation of the latter, allowing for the almost total dephosphorylation of peptides, (iii) a one-step enzymatic dephosphorylation, allowing for the partial dephosphorylation of the peptides and (iv) an additional endoGluC enzymatic hydrolysis, allowing for the cleavage of long-size peptides into shorter ones. The reverse-phase high-pressure liquid chromatography–tandem mass spectrometry (RP-HPLC-MS/MS) analyses of hydrolysates that underwent this four-phase strategy allowed for the identification of 28 phosphorylation sites (90%) out of the 31 referenced in UniprotKB/Swiss-Prot (1 June 2021), compared to 17 sites (54%) without the latter. The alpha-S2 casein phosphosites, referenced by their similarity in the UniProt database, were experimentally identified, whereas pSer148, pThr166 and pSer187 from a multiphosphorylated long-size kappa-casein were not. Data are available via ProteomeXchange with identifier PXD027132.

Identification of individual components of a commercial wheat germ acid phosphatase preparation.

Wheat germ acid phosphatase (WGAP) is a commercial preparation of partially purified protein commonly used in laboratory settings for non-specific enzymatic dephosphorylation. It is known that these preparations contain multiple phosphatase isozymes and are still relatively crude. This study therefore aimed to identify the protein components of a commercial preparation of wheat germ acid phosphatase using mass spectroscopy and comparative genomics. After one post-purchase purification step, the most prevalent fifteen proteins in the mixture included heat shock proteins, beta-amylases, glucoseribitol dehydrogenases, enolases, and an aminopeptidase. While not among the most abundant components, eight unique dephosphorylation enzymes were also present including three purple acid phosphatases. Furthermore, it is shown that some of these correspond to previously isolated isozymes; one of which has been also previously shown by transcriptome data to be overexpressed in wheat seeds. In summary, this study identified the major components of WGAP including phosphatases and hypothesizes the most active components towards a better understanding of this commonly used laboratory tool.

Preparation of pure lower-order myo-Inositol phosphates on laboratory scale for physiological and enzymatic studies

A simple method for the preparative production of lower-order myo-inositol phosphates was developed. Enzymatic phytate dephosphorylation was applied, because phytate-degrading enzymes generate usually predominantly one single myo-inositol phosphate isomer with five, four, three, two and one phosphate residue(s) bound to the myo-inositol ring in a regio- and stereoselective manner. The relative concentrations of the different lower-order myo-inositol phosphates in the reaction mixture were controlled by adjusting incubation time at 37 °C and a fixed phytate concentration and phytase activity. Purification of the individual lower-order myo-inositol phosphates was realized by anion-exchange chromatography on Q-Sepharose using a stepwise elution with ammonium formate:formic acid pH 2.5. Ethanol precipitation was successfully used to concentrate the pure lower-order myo-inositol phosphates. In a single approach 2–3 mg of pure myo-inositol tetrakis- or -trisphosphate isomers were obtained. About 60% of the initially applied phytate were converted into pure lower-order myo-inositol phosphates. The purified myo-inositol phosphate isomers were virtually free of other myo-inositol phosphate esters and could be used for enzymatic and physiological studies.

The phosphohistidine phosphatase SixA dephosphorylates the phosphocarrier NPr

Histidine phosphorylation is a posttranslational modification that alters protein function and also serves as an intermediate of phosphoryl transfer. Although phosphohistidine is relatively unstable, enzymatic dephosphorylation of this residue is apparently needed in some contexts, since both prokaryotic and eukaryotic phosphohistidine phosphatases have been reported. Here we identify the mechanism by which a bacterial phosphohistidine phosphatase dephosphorylates the nitrogen-related phosphotransferase system, a broadly conserved bacterial pathway that controls diverse metabolic processes. We show that the phosphatase SixA dephosphorylates the phosphocarrier protein NPr and that the reaction proceeds through phosphoryl transfer from a histidine on NPr to a histidine on SixA. In addition, we show that Escherichia coli lacking SixA are outcompeted by wild-type E. coli in the context of commensal colonization of the mouse intestine. Notably, this colonization defect requires NPr and is distinct from a previously identified in vitro growth defect associated with dysregulation of the nitrogen-related phosphotransferase system. The widespread conservation of SixA, and its coincidence with the phosphotransferase system studied here, suggests that this dephosphorylation mechanism may be conserved in other bacteria.

Limitations of an in vitro model of the poultry digestive tract on the evaluation of the catalytic performance of phytases.

The study aims to investigate the limitation of a poultry digestive tract model developed by Menezes-Blackburn et al. [J Agric Food Chem 63: 6142-6149 (2015)] on the evaluation of the bioefficacy of phytases. It was confirmed that the in vitro model does not mimic the in vivo situation in the birds sufficiently well to identify the best phytase product under real conditions, or to draw conclusion on the effect of phytate concentration, phytate source or feed composition on the bioefficacy of phytase. Addition of calcium ion (Ca2+ ) up to a concentration of 10 g kg-1 to the feed substrate, for example, did not affect enzymatic phytate dephosphorylation in the in vitro model in contrast to the observation in poultry. The in vitro approach was shown to be applicable as a complementary tool in the pre-selection of promising phytase candidates, resulting in a reduction in the number of feeding trials in the initial screening phase. © 2020 The Author. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Enzymatic Insertion of Lipids Increases Membrane Tension for Inhibiting Drug Resistant Cancer Cells.

Although lipids contribute to cancer drug resistance, it is challenging to target diverse range of lipids. Here, we show enzymatically inserting exceedingly simple synthetic lipids into membranes for increasing membrane tension and selectively inhibiting drug resistant cancer cells. The lipid, formed by conjugating dodecylamine to d-phosphotyrosine, self-assembles to form micelles. Enzymatic dephosphorylation of the micelles inserts the lipids into membranes and increases membrane tension. The micelles effectively inhibit a drug resistant glioblastoma cell (T98G) or a triple-negative breast cancer cell (HCC1937), without inducing acquired drug resistance. Moreover, the enzymatic reaction of the micelles promotes the accumulation of the lipids in the membranes of subcellular organelles (e.g., endoplasmic reticulum (ER), Golgi, and mitochondria), thus activating multiple regulated cell death pathways. This work, in which for the first time membrane tension is increased to inhibit cancer cells, illustrates a new and powerful supramolecular approach for antagonizing difficult drug targets.

Nucleotide-Selective Templated Self-Assembly of Nanoreactors under Dissipative Conditions.

Nature adopts complex chemical networks to finely tune biochemical processes. Indeed, small biomolecules play a key role in regulating the flux of metabolic pathways. Chemistry, which was traditionally focused on reactions in simple mixtures, is dedicating increasing attention to the network reactivity of highly complex synthetic systems, able to display new kinetic phenomena. Herein, we show that the addition of monophosphate nucleosides to a mixture of amphiphiles and reagents leads to the selective templated formation of self-assembled structures, which can accelerate a reaction between two hydrophobic reactants. The correct matching between nucleotide and the amphiphile head group is fundamental for the selective formation of the assemblies and for the consequent up-regulation of the chemical reaction. Transient stability of the nanoreactors is obtained under dissipative conditions, driven by enzymatic dephosphorylation of the templating nucleotides. These results show that small molecules can play a key role in modulating network reactivity, by selectively templating self-assembled structures that are able to up-regulate chemical reaction pathways.

Nucleotide‐Selective Templated Self‐Assembly of Nanoreactors under Dissipative Conditions

AbstractNature adopts complex chemical networks to finely tune biochemical processes. Indeed, small biomolecules play a key role in regulating the flux of metabolic pathways. Chemistry, which was traditionally focused on reactions in simple mixtures, is dedicating increasing attention to the network reactivity of highly complex synthetic systems, able to display new kinetic phenomena. Herein, we show that the addition of monophosphate nucleosides to a mixture of amphiphiles and reagents leads to the selective templated formation of self‐assembled structures, which can accelerate a reaction between two hydrophobic reactants. The correct matching between nucleotide and the amphiphile head group is fundamental for the selective formation of the assemblies and for the consequent up‐regulation of the chemical reaction. Transient stability of the nanoreactors is obtained under dissipative conditions, driven by enzymatic dephosphorylation of the templating nucleotides. These results show that small molecules can play a key role in modulating network reactivity, by selectively templating self‐assembled structures that are able to up‐regulate chemical reaction pathways.

Artificial Intracellular Filaments

Intracellular protein filaments are ubiquitous for cellular functions, but forming bona fide biomimetic intracellular filaments of small molecules in living cells remains elusive. Here, we report the in situ formation of self-limiting intracellular filaments of a small peptide via enzymatic morphological transition of a phosphorylated and trimethylated heterochiral tetrapeptide. Enzymatic dephosphorylation reduces repulsive intermolecular electrostatic interactions and converts the peptidic nanoparticles into filaments, which exhibit distinct types of cross-β structures with either C7 or C2 symmetries, with the hydrophilic C-terminal residues at the periphery of the helix. Macromolecular crowding promotes the peptide filaments to form bundles, which extend from the plasma membrane to nuclear membrane and hardly interact with endogenous components, including cytoskeletons. Stereochemistry and post-translational modification (PTM) of peptides are critical for generating the intracellular bundles. This work may offer a way to gain lost functions or to provide molecular insights for understanding normal and aberrant intracellular filaments.