39 research outputs found
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 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 dependent amidohydrolases that disfavour Znas 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 dependent isatin hydrolase like enzymes in comparison with the vast number of Zn dependent enzymes
Crystallization and molecular-replacement solution of a truncated form of human recombinant tetranectin
Tetranectin-Binding Site on Plasminogen Kringle 4 Involves the Lysine-Binding Pocket and at Least One Additional Amino Acid Residue
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, O<sub>2</sub>(a<sup>1</sup>Î<sub>g</sub>), is a ROS known to have
a wide range of effects on cell function. Nevertheless, mechanistic
details of singlet oxygenâs 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, O<sub>2</sub>(a<sup>1</sup>Î<sub>g</sub>). 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 <sup>3</sup>FMN
by ground state oxygen, O<sub>2</sub>(X<sup>3</sup>ÎŁ<sub>g</sub><sup>â</sup>), increases âź10-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 O<sub>2</sub>(a<sup>1</sup>Î<sub>g</sub>) formation for both SOPP and miniSOG.
We also show that the quantum yields of O<sub>2</sub>(a<sup>1</sup>Î<sub>g</sub>) production by both miniSOG and SOPP are mainly
limited by the fraction of FMN triplet states quenched by O<sub>2</sub>(X<sup>3</sup>ÎŁ<sub>g</sub><sup>â</sup>). The results
presented herein provide a much-needed quantitative framework that
will facilitate the future development of optogenetic ROS sensitizers
Tetranectin-Binding Site on Plasminogen Kringle 4 Involves the Lysine-Binding Pocket and at Least One Additional Amino Acid Residue â
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, O<sub>2</sub>(a<sup>1</sup>Î<sub>g</sub>),
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 O<sub>2</sub>(a<sup>1</sup>Î<sub>g</sub>) production in or on a cell.
As such, there is great need for a system that (a) selectively produces
O<sub>2</sub>(a<sup>1</sup>Î<sub>g</sub>) 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 O<sub>2</sub>(a<sup>1</sup>Î<sub>g</sub>) production.
As a consequence, our protein-encased FMN system produces O<sub>2</sub>(a<sup>1</sup>Î<sub>g</sub>) with the uniquely large quantum
efficiency of 0.25 Âą 0.03. We have also quantified other key
photophysical parameters that characterize this sensitizer system,
including unprecedented H<sub>2</sub>O/D<sub>2</sub>O solvent isotope
effects on the O<sub>2</sub>(a<sup>1</sup>Î<sub>g</sub>) formation
kinetics and yields. As such, our results facilitate future systematic
developments in this field