Tetranectin Binds to the Kringle 1-4 Form of Angiostatin and Modifies Its Functional Activity

Tetranectin is a plasminogen kringle 4 domain-binding protein present in plasma and various tissue locations. Decreased plasma tetranectin or increased tetranectin in stroma of cancers correlates with cancer progression and adverse prognosis. A possible mechanism through which tetranectin could influence cancer progression is by altering activities of plasminogen or the plasminogen fragment, angiostatin. Tetranectin was found to bind to the kringle 1-4 form of angiostatin (AST(K1-4)). In addition, tetranectin inhibited binding of plasminogen or AST(K1-4) to extracellular matrix (ECM) deposited by endothelial cells. Finally, tetranectin partially counteracted the ability of AST(K1-4) to inhibit proliferation of endothelial cells. This latter effect of tetranectin was specific for AST(K1-4) since it did not counteract the antiproliferative activities of the kringle 1-3 form of angiostatin (AST(K1-3)) or endostatin. These findings suggest that tetranectin may modulate angiogenesis through interactions with AST

A fundamental catalytic difference between zinc and manganese dependent enzymes revealed in a bacterial isatin hydrolase

The catalytic mechanism of the cyclic amidohydrolase isatin hydrolase depends on a catalytically active manganese in the substrate-binding pocket. The Mn$^{2+}$ ion is bound by a motif also present in other metal dependent hydrolases like the bacterial kynurenine formamidase. The crystal structures of the isatin hydrolases from Labrenzia aggregata and Ralstonia solanacearum combined with activity assays allow for the identification of key determinants specific for the reaction mechanism. Active site residues central to the hydrolytic mechanism include a novel catalytic triad Asp-His-His supported by structural comparison and hybrid quantum mechanics/classical mechanics simulations. A hydrolytic mechanism for a Mn$^{2+}$ dependent amidohydrolases that disfavour Zn$^{2+}$as the primary catalytically active site metal proposed here is supported by these likely cases of convergent evolution. The work illustrates a fundamental difference in the substrate-binding mode between Mn$^{2+}$ dependent isatin hydrolase like enzymes in comparison with the vast number of Zn$^{2+}$ dependent enzymes

No Photon Wasted: An Efficient and Selective Singlet Oxygen Photosensitizing Protein

Optogenetics has been, and will continue to be, a boon to mechanistic studies of cellular processes. Genetically encodable proteins that sensitize the production of reactive oxygen species (ROS) are expected to play an increasingly important role, particularly in elucidating mechanisms of temporally and spatially dependent cell signaling. However, a substantial challenge in developing such photosensitizing proteins has been to funnel the optical excitation energy into the initial selective production of only one ROS. Singlet molecular oxygen, O₂(a¹Δ_g), is a ROS known to have a wide range of effects on cell function. Nevertheless, mechanistic details of singlet oxygen’s behavior in a cell are lacking. On the basis of the rational optimization of a LOV-derived flavoprotein, we now report the development and photophysical characterization of a protein-encased photosensitizer that efficiently and selectively produces singlet oxygen at the expense of other ROS, especially ROS that derive from photoinduced electron transfer reactions. These results set the stage for a plethora of new experiments to elucidate ROS-mediated events in cells

Temperature Sensitive Singlet Oxygen Photosensitization by LOV-Derived Fluorescent Flavoproteins

Optogenetic sensitizers that selectively produce a given reactive oxygen species (ROS) constitute a promising tool for studying cell signaling processes with high levels of spatiotemporal control. However, to harness the full potential of this tool for live cell studies, the photophysics of currently available systems need to be explored further and optimized. Of particular interest in this regard, are the flavoproteins miniSOG and SOPP, both of which (1) contain the chromophore flavin mononucleotide, FMN, in a LOV-derived protein enclosure, and (2) photosensitize the production of singlet oxygen, O₂(a¹Δ_g). Here we present an extensive experimental study of the singlet and triplet state photophysics of FMN in SOPP and miniSOG over a physiologically relevant temperature range. Although changes in temperature only affect the singlet excited state photophysics slightly, the processes that influence the deactivation of the triplet excited state are more sensitive to temperature. Most notably, for both proteins, the rate constant for quenching of ³FMN by ground state oxygen, O₂(X³Σ_g^–), increases ∼10-fold upon increasing the temperature from 10 to 43 °C, while the oxygen-independent channels of triplet state deactivation are less affected. As a consequence, this increase in temperature results in higher yields of O₂(a¹Δ_g) formation for both SOPP and miniSOG. We also show that the quantum yields of O₂(a¹Δ_g) production by both miniSOG and SOPP are mainly limited by the fraction of FMN triplet states quenched by O₂(X³Σ_g^–). The results presented herein provide a much-needed quantitative framework that will facilitate the future development of optogenetic ROS sensitizers

A Proton Wire and Water Channel Revealed in the Crystal Structure of Isatin Hydrolase

The high resolution crystal structures of isatin hydrolase from Labrenzia aggregata in the apo and the product state are described. These are the first structures of a functionally characterized metal-dependent hydrolase of this fold. Isatin hydrolase converts isatin to isatinate and belongs to a novel family of metalloenzymes that include the bacterial kynurenine formamidase. The product state, mimicked by bound thioisatinate, reveals a water molecule that bridges the thioisatinate to a proton wire in an adjacent water channel and thus allows the proton released by the reaction to escape only when the product is formed. The functional proton wire present in isatin hydrolase isoform b represents a unique catalytic feature common to all hydrolases is here trapped and visualized for the first time. The local molecular environment required to coordinate thioisatinate allows stronger and more confident identification of orthologous genes encoding isatin hydrolases within the prokaryotic kingdom. The isatin hydrolase orthologues found in human gut bacteria raise the question as to whether the indole-3-acetic acid degradation pathway is present in human gut flora

Rational Design of an Efficient, Genetically Encodable, Protein-Encased Singlet Oxygen Photosensitizer

Singlet oxygen, O₂(a¹Δ_g), plays a key role in many processes of cell signaling. Limitations in mechanistic studies of such processes are generally associated with the difficulty of controlling the amount and location of O₂(a¹Δ_g) production in or on a cell. As such, there is great need for a system that (a) selectively produces O₂(a¹Δ_g) in appreciable and accurately quantifiable yields and (b) can be localized in a specific place at the suborganelle level. A genetically encodable, protein-encased photosensitizer is one way to achieve this goal. Through a systematic and rational approach involving mutations to a LOV2 protein that binds the chromophore flavin mononucleotide (FMN), we have developed a promising photosensitizer that overcomes many of the problems that affect related systems currently in use. Specifically, by decreasing the extent of hydrogen bonding between FMN and a specific amino acid residue in the local protein environment, we decrease the susceptibility of FMN to undesired photoinitiated electron-transfer reactions that kinetically compete with O₂(a¹Δ_g) production. As a consequence, our protein-encased FMN system produces O₂(a¹Δ_g) with the uniquely large quantum efficiency of 0.25 ± 0.03. We have also quantified other key photophysical parameters that characterize this sensitizer system, including unprecedented H₂O/D₂O solvent isotope effects on the O₂(a¹Δ_g) formation kinetics and yields. As such, our results facilitate future systematic developments in this field