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Proteomic interaction profiling reveals KIFC1 as a factor involved in early targeting of F508del-CFTR to degradation

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Abstract

Misfolded F508del-CFTR, the main molecular cause of the recessive disorder cystic fibrosis, is recognized by the endoplasmic reticulum (ER) quality control (ERQC) resulting in its retention and early degradation. The ERQC mechanisms rely mainly on molecular chaperones and on sorting motifs, whose presence and exposure determine CFTR retention or exit through the secretory pathway. Arginine-framed tripeptides (AFTs) are ER retention motifs shown to modulate CFTR retention. However, the interactions and regulatory pathways involved in this process are still largely unknown. Here, we used proteomic interaction profiling and global bioinformatic analysis to identify factors that interact differentially with F508del-CFTR and F508del-CFTR without AFTs (F508del-4RK-CFTR) as putative regulators of this specific ERQC checkpoint. Using LC–MS/MS, we identified kinesin family member C1 (KIFC1) as a stronger interactor with F508del-CFTR versus F508del-4RK-CFTR. We further validated this interaction showing that decreasing KIFC1 levels or activity stabilizes the immature form of F508del-CFTR by reducing its degradation. We conclude that the current approach is able to identify novel putative therapeutic targets that can be ultimately used to the benefit of CF patients.

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Notes

  1. R program, https://www.r-project.org/ (Last accessed March 09, 2018).

  2. GSEA database, http://software.broadinstitute.org/gsea/index.jsp (Last accessed February 18, 2017).

  3. DAVID database, https://david.ncifcrf.gov/ (Last accessed March 08, 2018).

  4. Reactome Pathway database, https://reactome.org/ (Last accessed September 30, 2017).

  5. APID database, http://cicblade.dep.usal.es:8080/APID/init.action (Last accessed January 26, 2018).

  6. Cytoscape, http://cytoscape.org (Last accessed January 26, 2018).

  7. Harvard PrimerBank database, https://pga.mgh.harvard.edu/primerbank/ (Last accessed November 06, 2017).

Abbreviations

ABC:

ATP-binding cassette

AFTs:

Arginine-framed tripeptides

APID:

Agile protein interactome dataserved

ATP:

Adenosine triphosphate

BHK:

Baby hamster kidney

BP:

Biologic process

cAMP:

cyclic adenosine monophosphate

CC:

Cellular compartment

CFBE41o-:

Cystic fibrosis bronchial epithelial cells

CF:

Cystic fibrosis

CFTR:

Cystic fibrosis transmembrane conductance regulator

DAVID:

Database for annotation: visualization and integrated discovery

DMP:

Dimethyl pimelimidate.2 HCl

DSP:

Dithiobis(succinimidylpropinate)

EMT:

Epithelial-to-mesenchymal transition

ENaC:

Epithelial Na+ channel

ER:

Endoplasmic reticulum

ES:

Enrichment score

ERQC:

Endoplasmic reticulum quality control

GSEA:

Gene set enrichment analysis

GO:

Gene ontology

HDACi:

Histone deacetylase inhibitor

HEK293:

Human embryonic kidney 293

iBAQ:

Intensity-based absolute quantification

MSD:

Membrane-spanning domain

NBD:

Nucleotide-binding domain

NT:

Non-treated

PM:

Plasma membrane

PPIs:

Protein–protein interactions

QC:

Quality control

RD:

Regulatory domain

SB:

Sample buffer

SEM:

Standard error of the mean

SUMO:

Small ubiquitin-like modifiers

TM:

Transmembrane segment

UPR:

Unfolded protein response

WCL:

Whole-cell lysate

Wt:

Wild-type

16HBE14o-:

Human bronchial epithelial cells

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Acknowledgements

Work supported by centre Grant UID/MULTI/04046/2013 to BioISI, Romain Pauwels Research Award to C.M.F, and Project MIMED PTDC/EEI-ESS/4923/2014 to A.O.F. SC and JDS are recipient of fellowships from BioSys PhD programme PD/BD/114393/2016 (SFRH/BD/52491/2014) and PD/BD/106084/2015 (SFRH(BD/106084/2015) from FCT, Portugal, respectively. Proteomics Core Facility-SGIKER is part of ProteoRed-ISCIII network and is partially funded by ERDF and ESF.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry & BioISI-Biosystems and Integrative Sciences Institute, Faculty of Sciences, University of Lisboa, Campo Grande, 1749-016, Lisbon, Portugal

    Sara Canato, João D. Santos, Margarida D. Amaral, André O. Falcao & Carlos M. Farinha

  2. CEDOC-Chronic Diseases Research Centre, Nova Medical School, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Rua Câmara Pestana, 1150-082, Lisbon, Portugal

    Ana S. Carvalho & Rune Matthiesen

  3. Proteomics Core Facility-SGIKER, University of the Basque Country UPV/EHU, Barrio Sariena, 48940, Vizcaya, Spain

    Kerman Aloria

  4. LASIGE, Faculty of Sciences, University of Lisboa, Campo Grande, 1749-016, Lisbon, Portugal

    André O. Falcao

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  1. Sara Canato
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  2. João D. Santos
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  3. Ana S. Carvalho
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  4. Kerman Aloria
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  5. Margarida D. Amaral
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  6. Rune Matthiesen
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  7. André O. Falcao
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  8. Carlos M. Farinha
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Contributions

SC designed and performed the experiments, analysed data and wrote the manuscript; JDS analysed data; ASC prepared the sample and performed mass spectrometry experiment; KA and RM performed the mass spectrometry experiment. MDA provided advice and comments on the manuscript; AOF guided the experiments design for bioinformatics analysis, secured funding and provided advice; CMF guided the project, secured funding, guided the experiments design and wrote the manuscript. All authors read, revised and approved the manuscript.

Corresponding author

Correspondence to Carlos M. Farinha.

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Fig. S1

- Characterization of CFBE cells stably expressing F508del-4RK-CFTR. CFBE cells expressing F508del-4RK-CFTR (lane 1-6), wt-CFTR (lane 7) and F508del-CFTR (lane 8) were grown at 37 °C or 26 °C and in some cases incubated with VX-809 (3 µM) for 24 h or DMSO (vehicle compound). Western blot (top) showing both mature form band C (~ 170 kDa) and immature form band B (~ 140 kDa) of CFTR protein. Calnexin (90 kDa) was used as a loading control (bottom). (JPEG 66 kb)

Fig. S2 - Bioinformatics analysis of the proteins differentially interacting with F508del-CFTR and F508del-4RK-CFTR. (A)

Hallmark enrichment for F508del-4RK-CFTR and F508del-CFTR using gene set enrichment (GSEA). GSEA was performed for a dataset of 834 proteins. Using GSEA tool, the subset of genes that contribute mostly to the enrichment of each hallmark was identified and plotted according to their affinity to F508del-4RK-CFTR (black dots) versus F508del-CFTR (grey dots) - represented by log2 of the ratio F508del-4RK-CFTR/F508del-CFTR. (B) Biological process represented for F508del-4RK-CFTR and F508del-CFTR using DAVID. From the total of 834 proteins, 198 with more affinity to F508del-CFTR and 164 proteins with more affinity to F508del-4RK-CFTR were used to find the GO terms – biological process enriched in both subsets. p value < 0.05. (C) Cellular component represented for F508del-4RK-CFTR and F508del-CFTR using DAVID. From the total of 834 proteins, 198 with more affinity to F508del-CFTR and 164 proteins with more affinity to F508del-4RK-CFTR were used to find the GO terms – cellular compartment enriched in both subsets. Significant levels are represented by p value < 0.05. (PDF 371 kb)

Fig. S3 - Scatter plot representing the differential protein interactions for the 22 putative hits in F508del-CFTR versus F508del-4RK-CFTR and peptides corresponding to the AFT regions of CFTR.

Log2 plot of the protein abundance ratio for F508del-4RK-CFTR versus F508del-CFTR and Log2 plot of the protein abundance ratio for peptide K (corresponding to mutated AFTs with Lys replacement) versus peptide R (Arg containing). Proteins were identified by LC–MS/MS and the threshold (log2 = ±1). (JPEG 113 kb)

Fig. S4

- Force-directed network of CFTR versus top hits interactome. Network comprises the connection of CFTR interactome (depicted in green circle) and the interactome of the selected hits: FKBP4, HNRNPA2B1, KIFC1, YWHAE (depicted in orange circles). All components comprising the interactors are predicted as nodes (yellow circles) and all were identified by LS-MS/MS covered the 834 proteins. Straight white lines illustrate edges that define interactions based on the APID protein interaction database which was accessed using Cytoscape platform. Dashed red lines illustrate the edges in which interactions occur with one node distance and full orange lines the edges in which interactions occur with two or more nodes distance to CFTR. (JPEG 149 kb)

Fig. S5 - Effect of FKBP4, 14-3-3ε and HNRNPA2B1 knockdown on F508del-CFTR protein processing.

CFBE cells expressing F508del-CFTR were transfected with siRNA against FKBP4, 14-3-3ε, HNRNPA2B1 or EGFP as non-targeting siRNA for 48 h. CT - transfection reagent only was used. Cells were also incubated with VX-809 (3 µM) or DMSO (vehicle control) are shown. Detection of F508del-CFTR protein expression (top) under (A) FKBP4, (B) 14-3-3ε or (C) HNRNPA2B1 knockdown. Equal amount of protein was loaded in each lane, as demonstrated by calnexin loading control (Bottom). (D) CFTR processing (band C divided by total CFTR) was normalized to siEGFP control. (E) Gene knockdown with siRNA in CFBE cells. Fold expression of FKBP4, 14-3-3ε or HNRNPA2B1 mRNA levels was obtained by relative quantification (ddCT method) and was normalized to an internal control (CAP-1). Data are shown as the mean ± SEM, n = 3. * p ≤ 0.05. Statistical analysis was performed using two-tailed unpaired Student’s t test. (JPEG 331 kb)

Fig. S6

- Effect of KIFC1 inhibition on F508del-CFTR expression. (A) CFBE cells expressing F508del-CFTR were incubated with 0.4 µM KIFC1 inhibitor (AZ82 compound) or DMSO, vehicle control (non-treated - NT), for 24 h, 48 h or 72 h. As a control for KIFC1 levels, cells were transfected with siRNA pool for KIFC1 or EGFP as non-targeting siRNA for 48 h. Western blot for CFTR (Top). Equal amount of protein was loaded in each lane, as demonstrated by α-tubulin (~ 50 kDa) detection (Bottom). (B) Quantification of CFTR (band B and C) band intensity normalized to loading control and to NT or siEGFP. (C) CFTR processing (band C divided by total CFTR) normalized to NT or siEGFP. Data are shown as mean ± SEM, n = 3. * ρ ≤ 0.05. Statistical analysis was performed using two-tailed unpaired Student’s t test. (JPEG 144 kb)

Fig. S7 - Distance of F508del-CFTR interactors to KIFC1.

The 198 interactors with higher affinity to F508del-CFTR were subjected to APID1 (accounting for all known interactions) and APID2 (accounting for interactions proved by two or more experiments). Each circle represents the distance from the proteins to KIFC1 (from one to five edges). Inside of each circle is represented the number of proteins showing the associated distance. (JPEG 92 kb)

Table S1 - Total interactome identified for F508del-CFTR and F508del-4RK-CFTR.

All identified proteins from MS analysis. (XLSX 156 kb)

Table S2 - Protein fold change interaction for F508del-CFTR versus F508del-4RK-CFTR.

Fold change interaction was obtained by log2 of the ratio of the amount of the protein detected in association with F508del-CFTR versus F508del-4RK-CFTR. (XLSX 94 kb)

Table S3 - Protein targets and inhibitors listed in ChEMBL database.

The 198 proteins with higher affinity for F508del-CFTR (log2 below -1) were searched in ChEMBL. The interactors with available inhibitors are listed along the fold change score (regarding F508del- over F508del-4RK-CFTR interaction). (XLSX 27 kb)

Table S4 - APID interactions linking CFTR and KIFC1.

The 198 proteins with stronger affinity with F508del-CFTR were analysed in APID1 and APID2 to identify their possible link to KIFC1, one to five represent the number of edges. (XLSX 30 kb)

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Canato, S., Santos, J.D., Carvalho, A.S. et al. Proteomic interaction profiling reveals KIFC1 as a factor involved in early targeting of F508del-CFTR to degradation. Cell. Mol. Life Sci. 75, 4495–4509 (2018). https://doi.org/10.1007/s00018-018-2896-7

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