6 research outputs found
Mutation of Nonessential Cysteines Shows That the NF-κB Essential Modulator Forms a Constitutive Noncovalent Dimer That Binds IκB Kinase‑β with High Affinity
NEMO
(NF-κB essential modulator) associates with catalytic
subunits IKKα and IKKβ to form the IκB kinase (IKK)
complex and is a key regulator of NF-κB pathway signaling. Biochemical
and structural characterization of NEMO has been challenging, however,
leading to conflicting data about basic biochemical properties such
as the oligomeric state of active NEMO and its binding affinity for
IKKβ. We show that up to seven of NEMO’s 11 cysteine
residues can be mutated to generate recombinant full-length NEMO that
is highly soluble and active. Using a fluorescence anisotropy binding
assay, we show that full-length NEMO binds a 44-mer peptide encompassing
residues 701–745 of IKKβ with a <i>K</i><sub>D</sub> of 2.2 ± 0.8 nM. The IKKβ binding affinities of
mutants with five and seven Cys-to-Ala substitutions are indistinguishable
from that of wild-type NEMO. Moreover, when expressed in NEMO −/–
fibroblasts, the five-Ala and seven-Ala NEMO mutants can interact
with cellular IKKβ and restore NF-κB signaling to provide
protection against tumor necrosis factor α-induced cell death.
Treatment of the NEMO-reconstituted cells with H<sub>2</sub>O<sub>2</sub> led to the formation of covalent dimers for wild-type NEMO
and the five-Ala mutant, but not for the seven-Ala mutant, confirming
that Cys54 and/or Cys347 can mediate interchain disulfide bonding.
However, the IKKβ binding affinity of NEMO is unaffected by
the presence or absence of interchain disulfide bonding at Cys54,
which lies within the IKKβ binding domain of NEMO, or at Cys347,
indicating that NEMO exists as a noncovalent dimer independent of
the redox state of its cysteines. This conclusion was corroborated
by the observation that the secondary structure content of NEMO and
its thermal stability were independent of the presence or absence
of interchain disulfide bonds
NF-κB Down-regulates Expression of the B-lymphoma Marker CD10 through a miR-155/PU.1 Pathway
Guidelines for the content and format of PET brain data in publications and archives : A consensus paper
It is a growing concern that outcomes of neuroimaging studies often cannot be replicated. To counteract this, the magnetic resonance (MR) neuroimaging community has promoted acquisition standards and created data sharing platforms, based on a consensus on how to organize and share MR neuroimaging data. Here, we take a similar approach to positron emission tomography (PET) data. To facilitate comparison of findings across studies, we first recommend publication standards for tracer characteristics, image acquisition, image preprocessing, and outcome estimation for PET neuroimaging data. The co-authors of this paper, representing more than 25 PET centers worldwide, voted to classify information as mandatory, recommended, or optional. Second, we describe a framework to facilitate data archiving and data sharing within and across centers. Because of the high cost of PET neuroimaging studies, sample sizes tend to be small and relatively few sites worldwide have the required multidisciplinary expertise to properly conduct and analyze PET studies. Data sharing will make it easier to combine datasets from different centers to achieve larger sample sizes and stronger statistical power to test hypotheses. The combining of datasets from different centers may be enhanced by adoption of a common set of best practices in data acquisition and analysis