58 research outputs found
The copper chaperone CCS facilitates copper binding to MEK1/2 to promote kinase activation
Normal physiology relies on the precise coordination of intracellular signaling pathways that respond to nutrient availability to balance cell growth and cell death. The canonical mitogen-activated protein kinase pathway consists of the RAFMEK- ERK signaling cascade and represents one of the most well-defined axes within eukaryotic cells to promote cell proliferation, which underscores its frequent mutational activation in human cancers. Our recent studies illuminated a function for the redox-active micronutrient copper (Cu) as an intracellular mediator of signaling by connecting Cu to the amplitude of mitogen-activated protein kinase signaling via a direct interaction between Cu and the kinases MEK1 and MEK2. Given the large quantities of molecules such as glutathione and metallothionein that limit cellular toxicity from free Cu ions, evolutionarily conserved Cu chaperones facilitate efficient delivery of Cu to cuproenzymes. Thus, a dedicated cellular delivery mechanism of Cu to MEK1/2 likely exists. Using surface plasmon resonance and proximity-dependent biotin ligase studies, we report here that the Cu chaperone for superoxide dismutase (CCS) selectively bound to and facilitated Cu transfer to MEK1. Mutants of CCS that disrupt Cu(I) acquisition and exchange or a CCS small-molecule inhibitor were used and resulted in reduced Cu-stimulated MEK1 kinase activity. Our findings indicate that the Cu chaperone CCS provides fidelity within a complex biological system to achieve appropriate installation of Cu within the MEK1 kinase active site that in turn modulates kinase activity and supports the development of novel MEK1/2 inhibitors that target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS
The copper centers of tyramine Ī²-monooxygenase and its catalytic-site methionine variants: an X-ray absorption study
Tyramine Ī²-monooxygenase (TBM) is a member of a family of copper monooxygenases containing two noncoupled copper centers, and includes peptidylglycine monooxygenase and dopamine Ī²-monooxygenase. In its Cu(II) form, TBM is coordinated by two to three His residues and one to two non-His O/N ligands consistent with a [CuM(His)2(OH2)2āCuH(His)3(OH2)] formulation. Reduction to the Cu(I) state causes a change in the X-ray absorption spectroscopy (XAS) spectrum, consistent with a change to a [CuM(His)2S(Met)āCuH(His)3] environment. Lowering the pH to 4.0 results in a large increase in the intensity of the Cu(I)āS extended X-ray absorption fine structure (EXAFS) component, suggesting a tighter CuāS bond or the coordination of an additional sulfurĀ donor. The XAS spectra of three variants, where the CuM Met471 residue had been mutated to His, Cys, and Asp, were examined. Significant differences from the wild-type enzyme are evident in the spectra of the reduced mutants. Although the side chains of His, Cys, and Asp are expected to substitute for Met at the CuM site, the data showed identical spectra for all three reduced variants, with no evidence for coordination of residue 471. Rather, the K-edge data suggested a modest decrease in coordination number, whereas the EXAFS indicated an average of two His residues at each Cu(I) center. These data highlight the unique role of the Met residue at the CuM center, and pose interesting questions as to why replacement by the cuprophilic thiolate ligand leads to detectable activity whereas replacement by imidazole generates inactive TBM
Characterization of a Half-Apo Derivative of Peptidylglycine Monooxygenase. Insight into the Reactivity of Each Active Site Copper ā
Does Superoxide Channel between the Copper Centers in Peptidylglycine Monooxygenase? A New Mechanism Based on Carbon Monoxide Reactivity ā
Identification of the cyanide stretching frequency in the cyano derivative of copper/zinc-superoxide dismutase by IR and Raman spectroscopy
Stable Cu(II) and Cu(I) Mononuclear Intermediates in the Assembly of the CuA Center of Thermus thermophilus Cytochrome Oxidase
CuA is a dinuclear mixed-valence center located in subunit
2 of
the <i>ba</i><sub>3</sub>-type cytochrome oxidase from Thermus thermophilus. The assembly of this site within
the periplasmic membrane is believed to be mediated by the copper
chaperones Sco and/or PCuAC, but the biological mechanisms are still
poorly understood, thereby stimulating interest in the mechanisms
of CuA formation from inorganic ions. The formulation of the CuA center
as an electron-delocalized Cu<sup>1.5</sup>āCu<sup>1.5</sup> system implicates both CuĀ(II) and CuĀ(I) states in the metalation
process. In earlier work we showed that selenomethionine (SeM) substitution
of the coordinated M160 residue provided a ligand-directed probe for
studying the copper coordination environment via the Se XAS signal,
which was particularly useful for interrogating the CuĀ(I) states where
other spectroscopic probes are absent. In the present study we have
investigated the formation of mixed-valence CuA and its M160SeM derivative
by stopped-flow UVāvis, EPR, and XAS at both Cu and Se edges,
while the formation of fully reduced di-CuĀ(I) CuA has been studied
by XAS alone. Our results establish the presence of previously undetected
mononuclear intermediates and show important differences from the
metalation reactions of purple CuA azurin. XAS spectroscopy at Cu
and Se edges has allowed us to extend mechanistic inferences to formation
of the di-CuĀ(I) state which may be more relevant to biological CuA
assembly. In particular, we find that T. thermophilus CuA assembles more rapidly than reported for other CuA systems and
that the dominant intermediate along the pathway to mixed-valence
is a new green species with Ī»<sub>max</sub> = 460 nm. This intermediate
has been isolated in a homogeneous state and shown to be a mononuclear
CuĀ(II)ā(His)Ā(Cys)<sub>2</sub> species with no observable CuĀ(II)ā(Met)
interaction. Reduction with dithionite generates its CuĀ(I) homologue
which is again mononuclear but now shows a strong interaction with
the Met160 thioether. The results are discussed within the framework
of the ācoupled distortionā model for CuĀ(II) thiolates
and their relevance to biological metalation reactions of the CuA
center
The Catalytic Role of the Copper Ligand H172 of Peptidylglycine Ī±-Hydroxylating Monooxygenase:Ā A Kinetic Study of the H172A Mutant ā
Spectroscopic Analysis of the Trinuclear Cluster in the Fet3 Protein from Yeast, a Multinuclear Copper Oxidase ā
Substrate-Induced Carbon Monoxide Reactivity Suggests Multiple Enzyme Conformations at the Catalytic Copper M-Center of Peptidylglycine Monooxygenase
Direct Metal Transfer between Periplasmic Proteins Identifies a Bacterial Copper Chaperone ā
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