Modulation of virus-induced NF-κB signaling by NEMO coiled coil mimics

Protein-protein interactions featuring intricate binding epitopes remain challenging targets for synthetic inhibitors. Interactions of NEMO, a scaffolding protein central to NF-κB signaling, exemplify this challenge. Various regulators are known to interact with different coiled coil regions of NEMO, but the topological complexity of this protein has limited inhibitor design. We undertook a comprehensive effort to block the interaction between vFLIP, a Kaposi’s sarcoma herpesviral oncoprotein, and NEMO using small molecule screening and rational design. Our efforts reveal that a tertiary protein structure mimic of NEMO is necessary for potent inhibition. The rationally designed mimic engages vFLIP directly causing complex disruption, protein degradation and suppression of NF-κB signaling in primary effusion lymphoma (PEL). NEMO mimic treatment induces cell death and delays tumor growth in a PEL xenograft model. Our studies with this inhibitor reveal the critical nexus of signaling complex stability in the regulation of NF-κB by a viral oncoprotein.


Statistics
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n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection TR-FRET data was collected using gen5 software , absorbance at 280nm was collected using FPLC system FPLC software, live confocal images were collected using ZEN microscope and imaging software, Flow cytometry data were collected using BD FACSDiVa™ software, bioluminescence images and quantification were collected using IVIS living image software Data analysis Microsoft excel 2010, Graphpad Prism version 6, FlowJo version 8.8.6, Fiji image processing package, Alphaspace 1.0 (http:// www.nyu.edu/projects/yzhang/AlphaSpace/), Rosetta computational alanine scanning For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Data generated or analyzed during this study are included in the published article (and its supplementary information files) or are available from the corresponding author upon reasonable request, see author contributions for specific data sets.

nature research | reporting summary
October 2018 Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
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Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
No statistical method was used to determine sample size before experiments were performed. Sample size was calculated based on estomated effect. Dat represent two or three independent experiments each performed in replciates. Single experiments were performed for assay types such as protein expression and purification,size exclusion chromatography

Validation
Validation statement for each antibody is proivided on the manufracturer's website. vFlip antibody was validated in non-PEL cell lines that are not infected with KSHV so they don't express the viral protein vFLIP. Flag antibody was validated in Namalwa cell line that is not induced with doxycycline . This cell line doesn't express the inducible flag-tagged vFLIP protein unless doxycline was added

Eukaryotic cell lines Policy information about cell lines
Cell line source(s) BC1, BC2 and BC3 PEL cell lines were established in Dr.Cesarman laboratory from lymphomatous effusions as described previously (cesarman, 1995, arvanitakis, 1996) , BCBL-1 was obtained from the AIDS and Cancer Specimen Bank. Namalwa Burkitt lymphoma cell line was purchased from American Type Culture Collection (ATCC). Stable WT vFLIP and mutant vFLIP inducible Namalwa cell lines were generated from the parental Namalwa cell line by lentiviral transduction. BC3-NF-kb luc cell line was generated from BC3 cell line using plasmid selection. HeLa cells were purchased from ATCC (lot# 70016358) nature research | reporting summary

Mycoplasma contamination
All cell lines were tested negative for mycoplasma contamination using IDEXX Bioresearch services or in the lab using PCR Mycoplasma Detection Kit ( Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.

Software
Flow cytometry data were collected using BD FACSDiVa™ software and data was analyzed using FlowJo version 8.8.6 Cell population abundancea Gating strategy A representative density plot with set gates is illustrated in figure 5C. Briefly, histogram with single stain compensation controls were used to distinguish positive and negative stains. Total population (live and dead cells) were gated on after excluding cellular debris based on their FSC-SSC distribution. Bivariate pseudocolor plot (Annexin-Alexa647, DAPI-Pacific blue) using single stain compensation controls was used to set the gates and the same gate was applied to all sample conditions.
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information. 0.5 million cells were stained for each condition and 300,000 to 500,000 events were collected from control and treated cells