Activation of Engineered Protein Tyrosine Phosphatases with the Biarsenical Compound AsCy3‐EDT2

Methods for activating signaling enzymes hold significant potential for the study of cellular signal transduction. Here we present a strategy for engineering chemically activatable protein tyrosine phosphatases (actPTPs). To generate actPTP1B, we introduced three cysteine point mutations in the enzyme's WPD loop. Biarsenical compounds were screened for the capability to bind actPTP1B's WPD loop and increase its phosphatase activity. We identified AsCy3‐EDT₂ as a robust activator that selectively targets actPTP1B in proteomic mixtures and intact cells. Introduction of the corresponding mutations in T‐cell PTP also generates an enzyme (actTCPTP) that is strongly activated by AsCy3‐EDT₂. Given the conservation of WPD‐loop structure among the classical PTPs, our results potentially provide the groundwork of a widely generalizable approach for generating actPTPs as tools for elucidating PTP signaling roles as well as connections between dysregulated PTP activity and human disease.     

Computational and Biochemical Design of a Nanopore Cleavable by a Cancer‐Secreted Enzyme

Many proteinaceous macromolecules selectively transport substrates across lipid bilayers and effectively serve as gated nanopores. Here, we engineered cleavage‐site motifs for human matrix metalloprotease 7 (MMP‐7) into the extracellular and pore‐constricting loops of OprD, a bacterial substrate‐specific transmembrane channel. Concurrent removal of two extracellular loops allowed MMP‐7 to access and hydrolyze a cleavage‐site motif engineered within the pore's major constricting loop, in both membrane‐incorporated and detergent‐solubilized OprDs. Import of antibiotics by the engineered OprDs into living bacteria pointed to their proper folding and integration in biological membranes. Purified engineered OprDs were also found to be properly folded in detergent. Hence, this study demonstrates the design of nanopores with a constriction cleavable by tumor‐secreted enzymes (like MMP‐7) for their potential incorporation in lipid‐based nanoparticles to accelerate drug release at the tumor site.     

Regulation of protein tyrosine phosphatase 1C: opposing effects of the two src homology 2 domains

The regulatory roles of the two src homology 2 (SH2) domainsof protein tyrosine phosphatase 1C were investigated by comparingrecombinant full-length PTP1C with mutants in which either theN-terminal SH2 (N-SH2) domain (PTP1CANSH2), the C-terminal SH2(C-SH2) domain (PTP1CACSH2) or both SH2 domains were deleted(PTP1CANSH2ACSH2). This revealed that the SH2 domains have opposingand independent effects on activity: strong inhibition by N-SH2(42-fold) and weak activation by C-SH2 (2.1-fold). C-SH2 causedactivation across a wide pH range while N-SH2 inhibited mostat neutral and high pH through a shift of the basic limb ofthe pH profile of kmt/Km, apparently via perturbation of anactive-site pKa value. A phosphotyrosyl peptide derived fromthe erythro-poietin receptor caused an {small tilde}30-foldactivation of PTP1C and PTP1CACSH2 but had no effect on PTP1CANSH2or PTP1CANSH2ACSH2, indicating that binding of this peptideto N-SH2 abolished its inhibition. Since C-SH2 separates N-SH2from the catalytic domain in full-length PTP1C and activationis observed for PTP1CACSH2, it appears that the inhibitory effectof N-SH2 is independent of the position in the sequence andthat intermolecular interactions may also be possible     

Light‐emitting polymers containing 2,3,4,5‐tetraphenylthiophenyl moiety and different pendent groups

By appropriate chemical reaction, different substituents can be selectively attached to the four phenyl rings present in 2,3,4,5‐tetraphenylthiophene (TP) to prepare monomers, namely 2,5‐bis(4‐bromophenyl)‐3,4‐diphenylthiophene (BTP), 2,5‐bis(4‐bromophenyl)‐3,4‐bis[4‐(nonan‐1‐one)phenyl] thiophene (BTP‐N2) and 2,5‐bis(4‐bromophenyl)‐3,4‐bis[4‐(2‐heptyl‐4‐phenylquinoline)phenyl]thiophene (BTP‐Qu2). Three light‐emitting polymers, PTP, PTP‐N2 and PTP‐Qu2, with the common TP backbone were prepared by zero‐valent nickel‐catalyzed polymerization of BTP, BTP‐N2 and BTP‐Qu2 monomers, respectively. The substituent on the 3,4‐phenyl rings of the TP framework has a profound effect on the polymer properties. Without any 3,4‐substituent, the rigid PTP polymer has low solubility in organic solvents. With the flexible nonanoyl substituent, the corresponding polymer, PTP‐N2, has improved solubility but low quantum efficiency (Φ_F) due to the carbonyl group which enhances intersystem crossing. With both flexible chain and bulky 4‐phenylquinoline (PQ) ring substituents, PTP‐Qu2 has good solubility and an enhanced Φ_F since the introduction of both flexible chain and bulky PQ ring substituents prevents close chain packing. All three polymers exhibit similar emission spectra despite the distinct difference in the absorption pattern of PTP‐Qu2 compared with those of PTP and PTP‐N2. In the case of PTP‐Qu2, there is energy transfer from the PQ pendent ring to the TP backbone and results in emission similar to PTP and PTP‐N2. The TP backbone common in all the three polymers is responsible for the emission from the corresponding excited states. The electrochemical properties of PTP‐Qu2 were also investigated. Copyright © 2005 Society of Chemical Industry     

A G-quadruplex aptamer inhibits the phosphatase activity of oncogenic protein Shp2 in vitro

Shp2 is a member of the protein tyrosine phosphatase (PTP) family, which regulates a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. Using a recombinant Shp2-GST protein as the target and GST as a counter target, we have identified two classes of single-stranded DNA aptamers that selectively bind to Shp2 with a K(d) in the nanomolar range. Structural studies of the most abundant sequence in the enriched library, HJ24, revealed a parallel G-quadruplex as the core binding domain. Furthermore, this aptamer was found to be an effective inhibitor of Shp2 phosphatase, an effect which was readily reversed by using the cDNA of HJ24. In view of these characteristics, this aptamer has the potential to be used for further development of Shp2 assays and therapeutics for the treatment of Shp2-dependent cancers and other diseases.     

An RNA Aptamer That Selectively Inhibits the Enzymatic Activity of Protein Tyrosine Phosphatase 1B in vitro

SELEX was used to create an RNA aptamer targeted to protein tyrosine phosphatase 1B (PTP1B), an enzyme implicated in type 2 diabetes, breast cancer and obesity. We found an aptamer that strongly inhibits PTP1B in vitro with a K_i of less than 600 pM . This slow‐binding, high‐affinity inhibitor is also highly selective, with no detectable effect on most other tested phosphatases and approximately 300:1 selectivity over the closely related TC‐PTP. Through controlled synthesis of truncated variants of the aptamer, we isolated shorter forms that inhibit PTP1B. We also investigated various single‐nucleotide modifications to probe their effects on the aptamer's secondary structure and inhibition properties. This family of aptamers represents an exciting option for the development of lead nucleotide‐based compounds in combating several human cancers and metabolic diseases.     

Effects of FlAsH/Tetracysteine (TC) Tag on PrP Proteolysis and PrPres Formation by TC‐Scanning

Protein–protein interactions associated with proteolytic processing and aggregation are integral to normal and pathological aspects of prion protein (PrP) biology. Characterization of these interactions requires the identification of amino acid residues involved. The FlAsH/tetracysteine (FlAsH/TC) tag is a small fluorescent tag amenable to insertion at internal sites in proteins. In this study, we used serial FlAsH/TC insertions (TC‐scanning) as a probe to characterize sites of protein–protein interaction between PrP and other molecules. To explore this application in the context of substrate–protease interactions, we analyzed the effect of FlAsH/TC insertions on proteolysis of cellular prion protein (PrPsen) in in vitro reactions and generation of the C1 metabolic fragment of PrPsen in live neuroblastoma cells. The influence of FlAsH/TC insertion was evaluated by TC‐scanning across the cleavage sites of each protease. The results showed that FlAsH/TC inhibited protease cleavage only within limited ranges of the cleavage sites, which varied from about one to six residues in width, depending on the protease, providing an estimate of the PrP residues interacting with each protease. TC‐scanning was also used to probe a different type of protein–protein interaction: the conformational conversion of FlAsH‐PrPsen to the prion disease‐associated isoform, PrPres. PrP constructs with FlAsH/TC insertions at residues 90–96 but not 97–101 were converted to FlAsH‐PrPres, identifying a boundary separating loosely versus compactly folded regions of PrPres. Our observations demonstrate that TC‐scanning with the FlAsH/TC tag can be a versatile method for probing protein–protein interactions and folding processes.     

Expanding Substrate Promiscuity by Engineering a Novel Adenylating‐Methylating NRPS Bifunctional Enzyme

Nonribosomal peptides synthetases (NRPSs), which are multifunctional mega‐enzymes producing many biologically active metabolites, are ideal targets for enzyme engineering. NRPS adenylation domains play a critical role in selecting/activating the amino acids to be transferred to downstream NRPS domains in the biosynthesis of natural products. Both monofunctional and bifunctional A domains interrupted with an auxiliary domain are found in nature. Here, we show that a bifunctional interrupted A domain can be uninterrupted by deleting its methyltransferase auxiliary domain portion to make an active monofunctional enzyme. We also demonstrate that a portion of an auxiliary domain with almost no sequence identity to the original auxiliary domain can be insert into naturally interrupted A domain to develop a new active bifunctional A domain with increased substrate profile. This work shows promise for the creation of new interrupted A domains in engineered NRPS enzymes.     

A Catalytic Nanoreactor Based on in Vivo Encapsulation of Multiple Enzymes in an Engineered Protein Nanocompartment

Bacterial protein compartments concentrate and sequester enzymes, thereby regulating biochemical reactions. Here, we generated a new functional nanocompartment in Escherichia coli by engineering the MS2 phage capsid protein to encapsulate multiple cargo proteins. Sequestration of multiple proteins in MS2‐based capsids was achieved by SpyTag/SpyCatcher protein fusions that covalently crosslinked with the interior surface of the capsid. Further, the functional two‐enzyme indigo biosynthetic pathway could be targeted to the engineered capsids, leading to a 60 % increase in indigo production in vivo. The enzyme‐loaded particles could be purified in their active form and showed enhanced long‐term stability in vitro (about 95 % activity after seven days) compared with free enzymes (about 5 % activity after seven days). In summary, this engineered in vivo encapsulation system provides a simple and versatile way for generating highly stable multi‐enzyme nanoreactors for in vivo and in vitro applications.     

Metabolic engineering of 2‐pentanone synthesis in Escherichia coli

Expanding the chemical diversity of microbial fermentation products enables green production of fuel, chemicals, and pharmaceuticals. In recent years, coenzyme A (CoA) dependent chain elongation, resembling the reversed β‐oxidation pathway, has attracted interest for its use in producing higher alcohols, fatty acids, and polyhydroxyalkanoate. To expand the chemical diversity of this pathway, we metabolically engineered Escherichia coli to produce 2‐pentanone, which is not a natural fermentation product of E. coli. We describe the first demonstration of 2‐pentanone synthesis in E. coli by coupling the CoA‐dependent chain elongation with the acetone production pathway. By bioprospecting for enzymes capable of efficient hydrolysis of 3‐keto‐hexanoyl‐CoA, production of 2‐pentanone increased 20 fold, reaching a titer of 240 mg/L. © 2013 American Institute of Chemical Engineers AIChE J, 59: 3167–3175, 2013     

Design of Hyperthermophilic Lipase Chimeras by Key Motif‐Directed Recombination

Recombination of diverse natural evolved domains within a superfamily offers greater opportunity for enzyme function leaps. How to recombine protein modules from distant parents with less disruption in cross‐interfaces is a challenging issue. Here, we identified the existence of a key motif, the sequence VVSVN(D)YR, within a structural motif ψ loop in the α/β‐hydrolase fold superfamily, by using a MEME server and the PROMOTIF program. To obtain thermostable lipase‐like enzymes, two chimeras were engineered at the key motif regions through recombination of domains from a mesophilic lipase and a hyperthermophilic esterase/peptidase with amino acid identity less than 21 %. The chimeras retained the desirable substrate preference of their mesophilic parent and exhibited more than 100‐fold increased thermostability at 50 °C. Through site‐directed mutation, we further improved activity of the chimera by 4.6‐fold. The recombination strategy presented here enables the creation of novel catalysts.     

Rational Design,Synthesis and Biological Evaluation of Modular Fluorogenic Substrates with High Affinity and Selectivity for PTP1B

Protein‐tyrosine phosphatase 1B (PTP1B) is a key regulatory enzyme in several signal transduction pathways, and its upregulation has been associated with type‐2 diabetes, obesity and cancer. Selective determination of the functional significance of PTP1B remains a major challenge because the activity of this crucial enzyme is currently evaluated through the use of fluorescent probes that lack selectivity and are limited to biochemical assays. Here we describe the rational design, synthesis and biological evaluation of new modular PTP1B fluorogenic substrates. The self‐immolative 4‐hydroxybenzyl alcohol has been used as a key component for the design of phosphotyrosine mimics linked to a latent chromophore, which is released through an enzyme‐initiated domino reaction. Preliminary biological investigations showed that, by optimising the stereoelectronic properties and the binding interactions at the enzyme active site, it is possible to achieve substrates with high affinity and promising selectivity. Due to their modular nature, the synthesised fluorogenic probes represent versatile tools; customisation of the different subunits could widen the scope of these probes to a broader range of in vitro assays. Finally, these studies elucidate the critical role played by Asp181 in the PTP1B‐catalysed dephosphorylation mechanism: disruption of the native conformation of this key amino acid residue on the WDP loop yields fluorogenic inhibitors, rather than substrates. For this reason, our studies also represent a step forward for the development of improved PTP1B noncovalent inhibitors.     

Selective Inhibitors of the Protein Tyrosine Phosphatase SHP2 Block Cellular Motility and Growth of Cancer Cells in vitro and in vivo

Selective inhibitors of the protein tyrosine phosphatase SHP2 (src homology region 2 domain phosphatase; PTPN11), an enzyme that is deregulated in numerous human tumors, were generated through a combination of chemical synthesis and structure‐based rational design. Seventy pyridazolon‐4‐ylidenehydrazinyl benzenesulfonates were prepared and evaluated in enzyme assays. The binding modes of active inhibitors were simulated in silico using a newly generated crystal structure of SHP2. The most powerful compound, GS‐493 (4‐{(2Z)‐2‐[1,3‐bis(4‐nitrophenyl)‐5‐oxo‐1,5‐dihydro‐4H‐pyrazol‐4‐yliden]hydrazino}benzenesulfonic acid; 25 ) inhibited SHP2 with an IC₅₀ value of 71±15 nM in the enzyme assay and was 29‐ and 45‐fold more active toward SHP2 than against related SHP1 and PTP1B. In cell culture experiments compound 25 was found to block hepatocyte growth factor (HGF)‐stimulated epithelial–mesenchymal transition of human pancreatic adenocarcinoma (HPAF) cells, as indicated by a decrease in the minimum neighbor distances of cells. Moreover, 25 inhibited cell colony formation in the non‐small‐cell lung cancer cell line LXFA 526L in soft agar. Finally, 25 was observed to inhibit tumor growth in a murine xenograft model. Therefore, the novel specific compound 25 strengthens the hypothesis that SHP2 is a relevant protein target for the inhibition of mobility and invasiveness of cancer cells.     

Structural Study of the Partially Disordered Full‐Length δ Subunit of RNA Polymerase from Bacillus subtilis

The partially disordered δ subunit of RNA polymerase was studied by various NMR techniques. The structure of the well‐folded N‐terminal domain was determined based on inter‐proton distances in NOESY spectra. The obtained structural model was compared to the previously determined structure of a truncated construct (lacking the C‐terminal domain). Only marginal differences were identified, thus indicating that the first structural model was not significantly compromised by the absence of the C‐terminal domain. Various ¹⁵N relaxation experiments were employed to describe the flexibility of both domains. The relaxation data revealed that the C‐terminal domain is more flexible, but its flexibility is not uniform. By using paramagnetic labels, transient contacts of the C‐terminal tail with the N‐terminal domain and with itself were identified. A propensity of the C‐terminal domain to form β‐type structures was obtained by chemical shift analysis. Comparison with the paramagnetic relaxation enhancement indicated a well‐balanced interplay of repulsive and attractive electrostatic interactions governing the conformational behavior of the C‐terminal domain. The results showed that the δ subunit consists of a well‐ordered N‐terminal domain and a flexible C‐terminal domain that exhibits a complex hierarchy of partial ordering.     

Activity of α‐Aminoadipate Reductase Depends on the N‐Terminally Extending Domain

L ‐α‐Aminoadipic acid reductases catalyze the ATP‐ and NADPH‐dependent reduction of L ‐α‐aminoadipic acid to the corresponding 6‐semialdehyde during fungal L ‐lysine biosynthesis. These reductases resemble peptide synthetases with regard to their multidomain composition but feature a unique domain of elusive function—now referred to as an adenylation activating (ADA) domain—that extends the reductase N‐terminally. Truncated enzymes based on NPS3, the L ‐α‐aminoadipic acid reductase of the basidiomycete Ceriporiopsis subvermispora, lacking the ADA domain either partially or entirely were tested for activity in vitro, together with an ADA‐adenylation didomain and the ADA domainless adenylation domain. We provide evidence that the ADA domain is required for substrate adenylation: that is, the initial step of the catalytic turnover. Our biochemical data are supported by in silico modeling that identified the ADA domain as a partial peptide synthetase condensation domain.     

Examination of a Structural Model of Peptidomimicry by Cyclic Acyldepsipeptide Antibiotics in Their Interaction with the ClpP Peptidase

The cyclic acyldepsipeptide (ADEP) antibiotics act by binding the ClpP peptidase and dysregulating its activity. Their exocyclic N‐acylphenylalanine is thought to structurally mimic the ClpP‐binding, (I/L)GF tripeptide loop of the peptidase's accessory ATPases. We found that ADEP analogues with exocyclic N‐acyl tripeptides or dipeptides resembling the (I/L)GF motif were weak ClpP activators and had no bioactivity. In contrast, ADEP analogues possessing difluorophenylalanine N‐capped with methyl‐branched acyl groups—like the side chains of residues in the (I/L)GF motifs—were superior to the parent ADEP with respect to both ClpP activation and bioactivity. We contend that the ADEP's N‐acylphenylalanine moiety is not simply a stand‐in for the ATPases' (I/L)GF motif; it likely has physicochemical properties that are better suited for ClpP binding. Further, our finding that the methyl‐branching on the acyl group of the ADEPs improves activity opens new avenues for optimization.     

Methods for stabilizing and activating enzymes in ionic liquids—a review

Ionic liquids (ILs) have evolved as a new type of non‐aqueous solvents for biocatalysis, mainly due to their unique and tunable physical properties. A number of recent review papers have described a variety of enzymatic reactions conducted in IL solutions; on the other hand, it is important to systematically analyze methods that have been developed for stabilizing and activating enzymes in ILs. This review discusses the biocatalysis in ILs from two unique aspects (1) factors that impact the enzyme's activity and stability, (2) methods that have been adopted or developed to activate and/or stabilize enzymes in ionic media. Factors that may influence the catalytic performance of enzymes include IL polarity, hydrogen‐bond basicity/anion nucleophilicity, IL network, ion kosmotropicity, viscosity, hydrophobicity, the enzyme dissolution, and surfactant effect. To improve the enzyme's activity and stability in ILs, major methods being explored include the enzyme immobilization (on solid support, sol–gel, or CLEA), physical or covalent attachment to PEG, rinsing with n‐propanol methods (PREP and EPRP), water‐in‐IL microemulsions, IL coating, and the design of enzyme‐compatible ionic solvents. It is exciting to notice that new ILs are being synthesized to be more compatible with enzymes. To utilize the full potential of ILs, it is necessary to further improve these methods for better enzyme compatibility. This is what has been accomplished in the field of biocatalysis in conventional organic solvents. Copyright © 2010 Society of Chemical Industry     

Dynamic substrate enhancement for the identification of specific, second-site-binding fragments targeting a set of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) are key regulators in living systems and thus are attractive drug targets. The development of potent, selective PTP inhibitors has been a difficult challenge mainly due to the high homology of the phosphotyrosine substrate pockets. Here, a strategy of dynamic substrate enhancement is described targeting the secondary binding sites of PTPs. By screening four different PTPs from bacterial (MptpA) and human origin (PTP1B, HePtp, Shp2) with this assay, specific fragments were identified. One highly specific fragment that binds to the secondary site of Mycobacterium tuberculosis protein tyrosine phosphatase A (MptpA) was characterized in order to validate the assay concept. Finally by covalently linking the secondary fragment to a phosphotyrosine mimetic, a moderately active but highly specific inhibitor of MptpA was obtained.     

Site‐Specific Immobilization of the Peptidoglycan Synthase PBP1B on a Surface Plasmon Resonance Chip Surface

Surface plasmon resonance (SPR) is one of the most powerful label‐free methods to determine the kinetic parameters of molecular interactions in real time and in a highly sensitive way. Penicillin‐binding proteins (PBPs) are peptidoglycan synthesis enzymes present in most bacteria. Established protocols to analyze interactions of PBPs by SPR involve immobilization to an ampicillin‐coated chip surface (a β‐lactam antibiotic mimicking its substrate), thereby forming a covalent complex with the PBPs transpeptidase (TP) active site. However, PBP interactions measured with a substrate‐bound TP domain potentially affect interactions near the TPase active site. Furthermore, in vivo PBPs are anchored in the inner membrane by an N‐terminal transmembrane helix, and hence immobilization at the C‐terminal TPase domain gives an orientation contrary to the in vivo situation. We designed a new procedure: immobilization of PBP by copper‐free click chemistry at an azide incorporated in the N terminus. In a proof‐of‐principle study, we immobilized Escherichia coli PBP1B on an SPR chip surface and used this for the analysis of the well‐characterized interaction of PBP1B with LpoB. The site‐specific incorporation of the azide affords control over protein orientation, thereby resulting in a homogeneous immobilization on the chip surface. This method can be used to study topology‐dependent interactions of any (membrane) protein.     

Superabsorbent hydrogel composed of covalently crosslinked gum arabic with fast swelling dynamics

A superabsorbent hydrogel (SH) with fast swelling dynamics and good mechanical stability in the swollen state was prepared. The SH was obtained from chemically modified gum arabic (MGA) with glycidyl methacrylate (GMA), acrylic acid (AAc), and acrylamide (AAm). The water‐transport mechanisms and equilibrium swelling ratio (SR) were the parameters used to describe the water‐absorption profile of SH. A 2³ factorial design with a central point was built to evaluate the influence of the chemical modification of gum arabic (GA) on the SH sensitivity to water. The main effects of the temperature and amount of GMA and the interaction effect of the temperature and amount of GMA on the SR responses were statistically significant. It was demonstrated that the amount of GMA had a more prominent effect on gel swelling. Lower elastic modulus values for the SH were found when a decreased amount of GMA was used (0.05 g), which produced an SH with a high SR capacity. The SH matrix consisting of 3.0 g of AAc, 0.5 g of AAm, and 0.5 g of MGA was more suitable to absorb a large amount of water (SR = 503.17 ± 22.65 within 60 min of water immersion) without its mechanical stability being affected. Better GA modifying conditions to produce such an SH were as follows: 60°C for the temperature, 24 h for the time of MGA, and 0.05 g for the amount of GMA. It exhibited a diffusional exponent higher than 0.89, which corresponded to the supercase II type of penetrating transport mechanism, indicating that its water‐absorption mechanism exclusively depended on the macromolecular relaxation of the polymer chains. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2008