Structural Insight into Substrate Selectivity of <i>Erwinia chrysanthemi</i> l‑Asparaginase

l-Asparaginases of bacterial origin are a mainstay of acute lymphoblastic leukemia treatment. The mechanism of action of these enzyme drugs is associated with their capacity to deplete the amino acid l-asparagine from the blood. However, clinical use of bacterial l-asparaginases is complicated by their dual l-asparaginase and l-glutaminase activities. The latter, even though representing only ∼10% of the overall activity, is partially responsible for the observed toxic side effects. Hence, l-asparaginases devoid of l-glutaminase activity hold potential as safer drugs. Understanding the key determinants of l-asparaginase substrate specificity is a prerequisite step toward the development of enzyme variants with reduced toxicity. Here we present crystal structures of the <i>Erwinia chrysanthemi</i> l-asparaginase in complex with l-aspartic acid and with l-glutamic acid. These structures reveal two enzyme conformationsopen and closedcorresponding to the inactive and active states, respectively. The binding of ligands induces the positioning of the catalytic Thr15 into its active conformation, which in turn allows for the ordering and closure of the flexible N-terminal loop. Notably, l-aspartic acid is more efficient than l-glutamic acid in inducing the active positioning of Thr15. Structural elements explaining the preference of the enzyme for l-asparagine over l-glutamine are discussed with guidance to the future development of more specific l-asparaginases

Structures of Apo and Product-Bound Human l‑Asparaginase: Insights into the Mechanism of Autoproteolysis and Substrate Hydrolysis

Asparaginases catalyze the hydrolysis of the amino acid asparagine to aspartate and ammonia. Bacterial asparaginases are used in cancer chemotherapy to deplete asparagine from the blood, because several hematological malignancies depend on extracellular asparagine for growth. To avoid the immune response against the bacterial enzymes, it would be beneficial to replace them with human asparaginases. However, unlike the bacterial asparaginases, the human enzymes have a millimolar <i>K</i>_m value for asparagine, making them inefficient in depleting the amino acid from blood. To facilitate the development of human variants suitable for therapeutic use, we determined the structure of human l-asparaginase (hASNase3). This asparaginase is an N-terminal nucleophile (Ntn) family member that requires autocleavage between Gly167 and Thr168 to become catalytically competent. For most Ntn hydrolases, this autoproteolytic activation occurs efficiently. In contrast, hASNas3 is relatively stable in its uncleaved state, and this allowed us to observe the structure of the enzyme prior to cleavage. To determine the structure of the cleaved state, we exploited our discovery that the free amino acid glycine promotes complete cleavage of hASNase3. Both enzyme states were elucidated in the absence and presence of the product aspartate. Together, these structures provide insight into the conformational changes required for cleavage and the precise enzyme–substrate interactions. The new understanding of hASNase3 will serve to guide the design of variants that possess a decreased <i>K</i>_m value for asparagine, making the human enzyme a suitable replacement for the bacterial asparaginases in cancer therapy

Targeted Delivery of Deoxycytidine Kinase to Her2-Positive Cells Enhances the Efficacy of the Nucleoside Analog Fludarabine

<div><p>Cytotoxic drugs, such as nucleoside analogs and toxins, commonly suffer from off-target effects. One approach to mitigate this problem is to deliver the cytotoxic drug selectively to the intended site. While for toxins this can be achieved by conjugating the cell-killing moiety to a targeting moiety, it is not an option for nucleoside analogs, which rely on intracellular enzymes to convert them to their active triphosphorylated form. To overcome this limitation, and achieve site-targeted activation of nucleoside analogs, we fused the coding region of a prodrug-activating enzyme, deoxycytidine kinase (dCK), to affinity reagents that bind to the Her2 cell surface protein. We evaluated dCK fusions to an anti-Her2 affibody and Designed Ankyrin Repeat Protein (DARPin) for their ability to kill cancer cells by promoting the activation of the nucleoside analog fludarabine. Cell staining and flow cytometry experiments with three Her2 positive cancer cell lines (BT-474-JB, JIMT-1 and SK-OV-3) indicate dCK fusions binding and cellular internalization. In contrast, these reagents bind only weakly to the Her2 negative cell line, MCF-7. Cell proliferation assays indicate that SK-OV-3 and BT-474-JB cell lines exhibit significantly reduced proliferation rates when treated with targeting-module fused dCK and fludarabine, compared to fludarabine alone. These findings demonstrate that we have succeeded in delivering active dCK into the Her2-positive cells, thereby increasing the activation of fludarabine, which ultimately reduces the dose of nucleoside analog needed for cell killing. This strategy may help establish the therapeutic index required to differentiate between healthy tissues and cancer cells.</p></div

Flexible and Transparent Triboelectric Nanogenerators Based on Polyoxometalate-Modified Polydimethylsiloxane Composite Films for Harvesting Biomechanical Energy

As one of the important friction materials for the construction of high-efficiency triboelectric nanogenerators (TENGs), polydimethylsiloxane (PDMS) has the advantages of high flexibility and transparency. However, the performance of pristine PDMS-based TENGs is not high enough, which limits its practical application. Polyoxometalates (POMs), as a class of nanoscale cluster compounds, have a strong ability to capture electrons. Appropriate POM materials can not only build nanostructures on the surface of PDMS without affecting its flexibility and transparency but also improve its surface roughness and enhance the ability to store charges, thereby enhancing the performance of TENGs. In this study, PDMS is modified by two kinds of Dawson-type POMs, and two POMs-TENGs are further constructed, named W-TENG and Mo-TENG, respectively. Performance tests show that the Mo-TENG exhibits an output voltage of 30 V and an output current of 500 nA, which are three times and twice that of the pristine PDMS-based TENG, respectively. This enhancement is attributed to POMs dispersed in the PDMS, which increase surface potential, surface roughness, and electronegativity. Finally, the application potential of Mo-TENG in wearable self-powered devices is demonstrated. This study expands the range of applications for POMs and provides an efficient and cost-effective method for the commercial manufacture of biosensors and self-powered devices

Strategy for preferential activation of prodrugs at target cells and the Her2-affinity reagents used in this study.

<p>(A) This strategy relies on a bi-modular fusion protein composed of a cell marker-targeting module (square labeled with T) genetically fused to an enzyme that catalyzes the activation of a prodrug (circle labeled with E). This fusion protein is administered systemically (point <b>1</b>), but it accumulates at the targeted cells by binding to a specific cell surface protein (point <b>2</b>). The fusion protein then enters the cell via receptor-mediated endocytosis or by membrane recycling (point <b>3</b>). Subsequent administration of an appropriate prodrug results in its preferential activation in the targeted cells (point <b>4</b>), thereby killing the targeted cell. (B) Ribbon diagram of the reagents with their molecular size indicated. Affibody, DARPin, and dCK models from PDB IDs 1LP1, 2JAB, and 1P5Z, respectively. The fusion proteins were modeled based on the individual structures. The dimeric nature of dCK result in molecules that contains two anti-Her2 modules, which are expected to increase its avidity to the receptor relative to the single affinity modules. Green spheres denote the substrates binding sites in dCK. (C) SDS-PAGE demonstrates the > 95% purity of the reagents. We note that the DARPin module runs as a smaller protein than the expected size. (D) Gel-filtration analysis of the reagents. The observed elution volumes correspond to the expected sizes of the reagents, with the fusion protein being dimeric, and the single affinity modules being monomeric.</p

Strategy for preferential activation of prodrugs at target cells and the Her2-affinity reagents used in this study.

<p>(A) This strategy relies on a bi-modular fusion protein composed of a cell marker-targeting module (square labeled with T) genetically fused to an enzyme that catalyzes the activation of a prodrug (circle labeled with E). This fusion protein is administered systemically (point <b>1</b>), but it accumulates at the targeted cells by binding to a specific cell surface protein (point <b>2</b>). The fusion protein then enters the cell via receptor-mediated endocytosis or by membrane recycling (point <b>3</b>). Subsequent administration of an appropriate prodrug results in its preferential activation in the targeted cells (point <b>4</b>), thereby killing the targeted cell. (B) Ribbon diagram of the reagents with their molecular size indicated. Affibody, DARPin, and dCK models from PDB IDs 1LP1, 2JAB, and 1P5Z, respectively. The fusion proteins were modeled based on the individual structures. The dimeric nature of dCK result in molecules that contains two anti-Her2 modules, which are expected to increase its avidity to the receptor relative to the single affinity modules. Green spheres denote the substrates binding sites in dCK. (C) SDS-PAGE demonstrates the > 95% purity of the reagents. We note that the DARPin module runs as a smaller protein than the expected size. (D) Gel-filtration analysis of the reagents. The observed elution volumes correspond to the expected sizes of the reagents, with the fusion protein being dimeric, and the single affinity modules being monomeric.</p

Effect of treatment of bi-modular fusion proteins on proliferation of cancer cells.

<p>(A) Seven thousand cells were treated with (i) buffer as control, (ii) bi-modular fusion protein (DARPin-dCK at 5 μM, panel A or (B) Affibody-dCK at 10 μM, panel B), (iii) fludarabine (0.25 μM), (iv) dCK (10 μM) and (v) as combination of fludarabine and bi-modular fusion protein at the same concentrations when tested by themselves. After the addition of the above reagents, cells were further incubated at 37°C for 96 h in 96 well plates, upon which cell proliferation was assessed using the AlamarBlue assay. The cell proliferation signal was normalized to the buffer control signal, which was set at 100%. Results are shown for the Her2-negative MCF-7 and Her2-positive BT-474 and SK-OV3 cell lines. Error bars correspond to standard deviations of triplicate measurements. p values were calculated using the Student’s T-test macros in Microsoft Excel (tail = 1, type = 3).</p

Characterizing individual anti-Her2 modules and the bi-modular anti-Her2-dCK fusion proteins.

<p>(A) Schematic of the ELISA assay used to evaluate the binding of the affinity reagents to the ectodomain IV of the Her2 receptor. (B) Binding of reagents to the ectodomain IV of Her2 receptor was measured by incubating 50 nM of reagents with 20 nM of immobilized Her2-Fc recombinant protein. Detection was performed using biotinylated anti-c-myc antibody and streptavidin conjugated to Horseradish peroxidase (HRP). Error bars represent standard deviation of triplicate measurements. We interpret the increased ELISA signal for the fusion proteins relative to the individual affinity modules resulting from the dimeric nature of the fusion proteins. (C) The observed steady state phosphorylation rate (k_obs) of fludarabine by the engineered dCK (dCK-DMS74E) with, or without, the Her2-affinity modules is measured with 200 μM of fludarabine at 37°C. These measurements indicate that the affinity modules do not interfere with the dCK enzymatic activity.</p

Binding of the bi-modular fusion proteins to cancer cells measured by flow cytometry.

<p>(A) One million cells were treated with 1 μM of reagents as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157114#pone.0157114.g002" target="_blank">Fig 2</a> and mean fluorescence intensity was measured using a Cyan3 fluorescent cell sorter (Beckman). Fluorescent cells were gated on cells treated with reagents that were not conjugated to dye and hence did not exhibit any fluorescence at 647 nm. Error bars correspond to standard deviations on three independent trials. (B) One million cells were treated with 1 μM dCK-AlexaFluor^™647 and the signal intensity was normalized to number of dye conjugations (ref 3 from Supplement). Error bars correspond to standard deviations of n = 3 trials. (C) Cell were treated with 2 μM of anti-Her2 DARPin-AlexaFluor^™647 and Affibody-AlexaFluor^™647 and measured as above. Note the much reduced mean fluorescence intensity in comparison to the fusion constructs (panel A). Error bars correspond to standard deviations of three independent trials. (D) Fluorescence intensities of cells treated with dCK-fusion protein were normalized to number of conjugated dye molecules (3), background subtracted and compared to those of cells treated with the Her2 affinity module (DARPin or affibody) alone. The resulting mean fold change in intensity was plotted for each cell type and reagent.</p

Exploring the Nature of Cellulose Microfibrils

Ultrathin cellulose microfibril fractions were extracted from spruce wood powder using combined delignification, TEMPO-catalyzed oxidation, and sonication processes. Small-angle X-ray scattering of these microfibril fractions in a “dilute” aqueous suspension (concentration 0.077 wt %) revealed that their shape was in the form of nanostrip with 4 nm width and only about 0.5 nm thicknesses. These dimensions were further confirmed by TEM and AFM measurements. The 0.5 nm thickness implied that the nanostrip could contain only a single layer of cellulose chains. At a higher concentration (0.15 wt %), SAXS analysis indicated that these nanostrips aggregated into a layered structure. The X-ray diffraction of samples collected at different preparation stages suggested that microfibrils were delaminated along the (11̅0) planes from the I_β cellulose crystals. The degree of oxidation and solid-state ¹³C NMR characterizations indicated that, in addition to the surface molecules, some inner molecules of microfibrils were also oxidized, facilitating the delamination into cellulose nanostrips