Role of asparaginase variable loop at the carboxyl terminal of the alpha subunit in the determination of substrate preference in plants.

Structural determinants responsible for the substrate preference of the potassium-independent (ASPGA1) and -dependent (ASPGB1) asparaginases from Arabidopsis thaliana have been investigated. Like ASPGA1, ASPGB1 was found to be catalytically active with both L: -Asn and ß-Asp-His as substrates, contrary to a previous report. However, ASPGB1 had a 45-fold higher specific activity with Asn as substrate than ASPGA1. A divergent sequence between the two enzymes forms a variable loop at the C-terminal of the alpha subunit. The results of dynamic simulations have previously implicated a movement of the C-terminus in the allosteric transduction of K(+)-binding at the surface of LjNSE1 asparaginase. In the crystal structure of Lupinus luteus asparaginase, most residues in this segment cannot be visualized due to a weak electron density. Exchanging the variable loop in ASPGA1 with that from ASPGB1 increased the affinity for Asn, with a 320-fold reduction in K (m) value. Homology modeling identified a residue specific to ASPGB1, Phe(162), preceding the variable loop, whose side chain is located in proximity to the beta-carboxylate group of the product aspartate, and to Gly(246), a residue participating in an oxyanion hole which stabilizes a negative charge forming on the side chain oxygen of asparagine during catalysis. Replacement with the corresponding leucine from ASPGA1 specifically lowered the V (max) value with Asn as substrate by 8.4-fold.

Application of Aspergillus niger Fumonisin Amine Oxidase (AnFAO) to Detoxify Fumonisin-Contaminated Maize.

Fumonisin mycotoxins are a family of secondary metabolites produced by Fusarium verticillioides and related species, as well as some strains of Aspergillus niger. Fumonisin contamination of maize is a concern when grown under hot, dry conditions. When present above regulatory levels, there can be effects on animal health. New tools to reduce the toxicity of maize and maize products with high concentrations of fumonisin are needed. Recently, we reported an amine oxidase (AnFAO) from a fumonisin-producing Aspergillus niger strain capable of oxidatively deaminating intact fumonisins. In this study, AnFAO was used to reduce intact fumonisin concentrations in milled maize flour, whole kernel maize inoculated with fumonisin-producing Fusarium verticillioides, and dried distillers' grains with solubles (DDGS). The data showed that milled maize flour incubated with 1 µM AnFAO for 1 h resulted in complete deamination of FB1 and FB2. A greater than 90% reduction in FB1-3 concentrations was observed following a simple washing procedure of whole kernel maize in the presence of 1 µM AnFAO for 1 h. Similarly, a ≥86% reduction in FB1-3 concentrations was observed in DDGS after 4 h incubation with 1 µM AnFAO. Finally, we engineered the methylotrophic yeast Pichia pastoris to produce functional AnFAO in both a secreted and intracellular form. These results support the further development and application of AnFAO as a promising tool to remediate fumonisin-contaminated maize and maize products.

RHAMM promotes interphase microtubule instability and mitotic spindle integrity through MEK1/ERK1/2 activity.

An oncogenic form of RHAMM (receptor for hyaluronan-mediated motility, mouse, amino acids 163-794 termed RHAMM(Delta163)) is a cell surface hyaluronan receptor and mitotic spindle protein that is highly expressed in aggressive human cancers. Its regulation of mitotic spindle integrity is thought to contribute to tumor progression, but the molecular mechanisms underlying this function have not previously been defined. Here, we report that intracellular RHAMM(Delta163) modifies the stability of interphase and mitotic spindle microtubules through ERK1/2 activity. RHAMM(-/-) mouse embryonic fibroblasts exhibit strongly acetylated interphase microtubules, multi-pole mitotic spindles, aberrant chromosome segregation, and inappropriate cytokinesis during mitosis. These defects are rescued by either expression of RHAMM or mutant active MEK1. Mutational analyses show that RHAMM(Delta163) binds to alpha- and beta-tubulin protein via a carboxyl-terminal leucine zipper, but in vitro analyses indicate this interaction does not directly contribute to tubulin polymerization/stability. Co-immunoprecipitation and pulldown assays reveal complexes of RHAMM(Delta163), ERK1/2-MEK1, and alpha- and beta-tubulin and demonstrate direct binding of RHAMM(Delta163) to ERK1 via a D-site motif. In vitro kinase analyses, expression of mutant RHAMM(Delta163) defective in ERK1 binding in mouse embryonic fibroblasts, and blocking MEK1 activity collectively confirm that the effect of RHAMM(Delta163) on interphase and mitotic spindle microtubules is mediated by ERK1/2 activity. Our results suggest a model wherein intracellular RHAMM(Delta163) functions as an adaptor protein to control microtubule polymerization during interphase and mitosis as a result of localizing ERK1/2-MEK1 complexes to their tubulin-associated substrates.

Identification and Characterization of an Aspergillus niger Amine Oxidase that Detoxifies Intact Fumonisins.

Fumonisin contamination of maize damaged by Fusarium verticillioides and related species is a major problem when it is grown under warm and dry conditions. Consumption of fumonisin contaminated food and feed is harmful to both humans and livestock. Novel tools for reducing or eliminating fumonisin toxicity may be useful to the agri-feed sector to deal with this worldwide problem. Enzymes capable of catabolizing fumonisins have been identified from microorganisms that utilize fumonisins as an energy source. However, fumonisin detoxifying enzymes produced by the very species that biosynthesize the toxin have yet to be reported. Here we describe the identification and characterization of a novel amine oxidase synthesized by the fumonisin-producing fungus Aspergillus niger. We have recombinantly expressed this A. niger enzyme in E. coli and demonstrated its ability to oxidatively deaminate intact fumonisins without requiring exogenous cofactors. This enzyme, termed AnFAO (A. niger fumonisin amine oxidase), displays robust fumonisin deamination activity across a broad range of conditions, has a high native melting temperature, and can be purified to >95% homogeneity at high yield in a one-step enrichment. AnFAO is a promising tool to remediate fumonisin-contaminated feed including maize destined for ethanol production.

Stimulation of the maltose transporter ATPase by unliganded maltose binding protein.

ATP hydrolysis by the maltose transporter (MalFGK(2)) is regulated by maltose binding protein (MBP). Binding of maltose to MBP brings about a conformational change from open to closed that leads to a strong stimulation of the MalFGK(2) ATPase. In this study, we address the long-standing but enigmatic observation that unliganded MBP is also able to stimulate MalFGK(2). Although the mechanism of this stimulation is not understood, it is sometimes attributed to a small amount of closed (but unliganded) MBP that may exist in solution. To gain insight into how MBP regulates the MalFGK(2) ATPase, we have investigated whether the open or the closed conformation of MBP is responsible for MalFGK(2) stimulation in the absence of maltose. The effect of MBP concentration on the stimulation of MalFGK(2) was assessed: for unliganded MBP, the apparent K(M) for stimulation of MalFGK(2) was below 1 microM, while for maltose-bound MBP, the K(M) was approximately 15 microM. We show that engineered MBP molecules in which the open-closed equilibrium has been shifted toward the closed conformation have a decreased ability to stimulate MalFGK(2). These results indicate that stimulation of the MalFGK(2) ATPase by unliganded MBP does not proceed through a closed conformation and instead must operate through a different mechanism than stimulation by liganded MBP. One possible explanation is that the open conformation is able to activate the MalFGK(2) ATPase directly.

Translational models of prostate cancer bone metastasis.

Metastatic disease is the principal cause of prostate-cancer-related mortality. Our ability to accurately recapitulate the spread of prostate cancer to bone - the most common site of metastasis - is critical to the development of novel metastasis-directed therapies. Several translational models of prostate cancer bone metastasis have been developed, including animal models, cell line injection models, 3D in vitro models, bone implant models, and patient-derived xenograft models. The use of these models has led to numerous advances in elucidating the molecular mechanisms of metastasis and innovations in targeted therapy. Despite this progress, current models are limited by a failure to holistically reproduce each individual element of the metastatic cascade in prostate cancer bone metastasis. In addition, factors such as accurate recapitulation of immunobiological events and improvements in tumour heterogeneity require further consideration. Knowledge gained from historical and currently used models will improve the development of next-generation models. An introspective appraisal of current preclinical models demonstrating bone metastases is warranted to narrow research focus, improve future translational modelling, and expedite the delivery of urgently needed metastasis-directed treatments.

Structural studies of an engineered zinc biosensor reveal an unanticipated mode of zinc binding.

Protein engineering was used previously to convert maltose-binding protein (MBP) into a zinc biosensor. Zn(2+) binding by the engineered MBP was thought to require a large conformational change from "open" to "closed", similar to that observed when maltose is bound by the wild-type protein. We show that although this re-designed MBP molecule binds Zn(2+) with high affinity as previously reported, it does not adopt a closed conformation in solution as assessed by small-angle X-ray scattering. High-resolution crystallographic studies of the engineered Zn(2+)-binding MBP molecule demonstrate that Zn(2+) is coordinated by residues on the N-terminal lobe only, and therefore Zn(2+) binding does not require the protein to adopt a fully closed conformation. Additional crystallographic studies indicate that this unexpected Zn(2+) binding site can also coordinate Cu(2+) and Ni(2+) with only subtle changes in the overall conformation of the protein. This work illustrates that the energetic barrier to domain closure, which normally functions to maintain MBP in an open concentration in the absence of ligand, is not easily overcome by protein design. A comparison to the mechanism of maltose-induced domain rearrangement is discussed.

Hyaluronan, Inflammation, and Breast Cancer Progression.

Breast cancer-induced inflammation in the tumor reactive stroma supports invasion and malignant progression and is contributed to by a variety of host cells including macrophages and fibroblasts. Inflammation appears to be initiated by tumor cells and surrounding host fibroblasts that secrete pro-inflammatory cytokines and chemokines and remodel the extracellular matrix (ECM) to create a pro-inflammatory "cancerized" or tumor reactive microenvironment that supports tumor expansion and invasion. The tissue polysaccharide hyaluronan (HA) is an example of an ECM component within the cancerized microenvironment that promotes breast cancer progression. Like many ECM molecules, the function of native high-molecular weight HA is altered by fragmentation, which is promoted by oxygen/nitrogen free radicals and release of hyaluronidases within the tumor microenvironment. HA fragments are pro-inflammatory and activate signaling pathways that promote survival, migration, and invasion within both tumor and host cells through binding to HA receptors such as CD44 and RHAMM/HMMR. In breast cancer, elevated HA in the peri-tumor stroma and increased HA receptor expression are prognostic for poor outcome and are associated with disease recurrence. This review addresses the critical issues regarding tumor-induced inflammation and its role in breast cancer progression focusing specifically on the changes in HA metabolism within tumor reactive stroma as a key factor in malignant progression.

How does a protein with dual mitotic spindle and extracellular matrix receptor functions affect tumor susceptibility and progression?

The mechanisms responsible for the oncogenic effects of the hyaluronan (HA) receptor and mitotic spindle binding protein, RHAMM, are poorly understood. On one hand, extracellular RHAMM interacts with HA and cellsurface receptors such as CD44 to coordinately activate the MAPK/ERK1,2 pathway, thus contributing to the spread and proliferation of tumor cells. On the other hand, intracellular RHAMM decorates mitotic spindles and is necessary for spindle formation and progression through G2/M and overexpression or loss of RHAMM can result in multipole spindles and chromosome missegregation. The deregulation of these intracellular functions could lead to genomic instability and fuel tumor progression. This suggests that both extracellular and intracellular RHAMM can promote tumor progression. Intracellular RHAMM can bind directly to ERK1 to form complexes with ERK2, MEK1 and ERK1,2 substrates, and we present a model whereby RHAMM's function is as a scaffold protein, controlling activation and targeting of ERK1,2 to specific substrates.

Insights into the conformational equilibria of maltose-binding protein by analysis of high affinity mutants.

The affinity of maltose-binding protein (MBP) for maltose and related carbohydrates was greatly increased by removal of groups in the interface opposite the ligand binding cleft. The wild-type protein has a KD of 1200 nM for maltose; mutation of residues Met-321 and Gln-325, both to alanine, resulted in a KD for maltose of 70 nM; deletion of 4 residues, Glu-172, Asn-173, Lys-175, and Tyr-176, which are part of a poorly ordered loop, results in a KD for maltose of 110 nM. Combining the mutations yields an increased affinity for maltodextrins and a KD of 6 nM for maltotriose. Comparison of ligand binding by the mutants, using surface plasmon resonance spectroscopy, indicates that decreases in the off-rate are responsible for the increased affinity. Small-angle x-ray scattering was used to demonstrate that the mutations do not significantly affect the solution conformation of MBP in either the presence or absence of maltose. The crystal structures of selected mutants showed that the mutations do not cause significant structural changes in either the closed or open conformation of MBP. These studies show that interactions in the interface opposite the ligand binding cleft, which we term the "balancing interface," are responsible for modulating the affinity of MBP for its ligand. Our results are consistent with a model in which the ligand-bound protein alternates between the closed and open conformations, and removal of interactions in the balancing interface decreases the stability of the open conformation, without affecting the closed conformation.