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Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride and bicarbonate channel in secretory epithelia with a critical role in maintaining fluid homeostasis. Mutations in CFTR are associated with Cystic Fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasians. While remarkable treatment advances have been made recently in the form of modulator drugs directly rescuing CFTR dysfunction, there is still considerable scope for improvement of therapeutic effectiveness. Here, we report the application of a high-throughput screening variant of the Mammalian Membrane Two-Hybrid (MaMTH-HTS) to map the protein–protein interactions of wild-type (wt) and mutant CFTR (F508del), in an effort to better understand CF cellular effects and identify new drug targets for patient-specific treatments. Combined with functional validation in multiple disease models, we have uncovered candidate proteins with potential roles in CFTR function/CF pathophysiology, including Fibrinogen Like 2 (FGL2), which we demonstrate in patient-derived intestinal organoids has a significant effect on CFTR functional expression.

Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes., QC 20200304

Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes.

Contains fulltext : 182332.pdf (publisher's version ) (Open Access), Regulation of our water homeostasis is fine-tuned by dynamic translocation of Aquaporin-2 (AQP2)-bearing vesicles to and from the plasma membrane of renal principal cells. Whereas binding of vasopressin to its type-2 receptor initiates a cAMP-protein kinase A cascade and AQP2 translocation to the apical membrane, this is counteracted by protein kinase C-activating hormones, resulting in ubiquitination-dependent internalization of AQP2. The proteins targeting AQP2 for ubiquitin-mediated degradation are unknown. In collecting duct mpkCCD cells, siRNA knockdown of NEDD4 and NEDD4L E3 ligases yielded increased AQP2 abundance, but they did not bind AQP2. Membrane Yeast Two-Hybrid assays using full-length AQP2 as bait, identified NEDD4 family interacting protein 2 (NDFIP2) to bind AQP2. NDFIP2 and its homologue NDFIP1 have PY motifs by which they bind NEDD4 family members and bring them close to target proteins. In HEK293 cells, NDFIP1 and NDFIP2 bound AQP2 and were essential for NEDD4/NEDD4L-mediated ubiquitination and degradation of AQP2, an effect not observed with PY-lacking NDFIP1/2 proteins. In mpkCCD cells, downregulation of NDFIP1, NEDD4 and NEDD4L, but not NDFIP2, increased AQP2 abundance. In mouse kidney, Ndfip1 and Ndfip2 mRNA distribution was similar and high in proximal tubules and collecting ducts, which was also found for NDFIP1 proteins. Our results reveal that NEDD4/NEDD4L mediate ubiquitination and degradation of AQP2, but that NDFIP proteins are needed to connect NEDD4/NEDD4L to AQP2. As NDFIP1/2 bind many NEDD4 family E3 ligases, which are implicated in several cellular processes, NDFIP1/2 may be the missing link for AQP2 ubiquitination and degradation from different subcellular locations.

RECQL4 belongs to the conserved RecQ family of DNA helicases, members of which play important roles in the maintenance of genome stability in all organisms that have been examined. Although genetic alterations in the RECQL4 gene are reported to be associated with three autosomal recessive disorders (Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes), the molecular role of RECQL4 still remains poorly understood. Here, we show that RECQL4 specifically interacts with the histone acetyltransferase p300 (also known as p300 HAT), both in vivo and in vitro, and that p300 acetylates one or more of the lysine residues at positions 376, 380, 382, 385 and 386 of RECQL4. Furthermore, we report that these five lysine residues lie within a short motif of 30 amino acids that is essential for the nuclear localization of RECQL4. Remarkably, the acetylation of RECQL4 by p300 in vivo leads to a significant shift of a proportion of RECQL4 protein from the nucleus to the cytoplasm. This accumulation of the acetylated RECQL4 is a result of its inability to be imported into the nucleus. Our results provide the first evidence of a post-translational modification of the RECQL4 protein, and suggest that acetylation of RECQL4 by p300 regulates the trafficking of RECQL4 between the nucleus and the cytoplasm.

Bloom's syndrome (BS) is a cancer predisposition disorder caused by mutation of the BLM gene, encoding a member of the RecQ helicase family. Although the phenotype of BS cells is suggestive of a role for BLM in repair of stalled or damaged replication forks, thus far there has been no direct evidence that BLM associates with any of the three human replicative DNA polymerases. Here, we show that BLM interacts specifically in vitro and in vivo with p12, the smallest subunit of human POL {delta} (hPOL {delta}). The hPOL {delta} enzyme, as well as the isolated p12 subunit, stimulates the DNA helicase activity of BLM. Conversely, BLM stimulates hPOL {delta} strand displacement activity. Our results provide the first functional link between BLM and the replicative machinery in human cells, and suggest that BLM might be recruited to sites of disrupted replication through an interaction with hPOL {delta}. Finally, our data also define a novel role for the poorly characterized p12 subunit of hPOL {delta}.

PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this "MYTH 2.0" system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPi-IIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes.

Werner syndrome (WS) is a severe recessive disorder characterized by premature aging, cancer predisposition and genomic instability. The gene mutated in WS encodes a bi-functional enzyme called WRN that acts as a RecQ-type DNA helicase and a 3'-5' exonuclease, but its exact role in DNA metabolism is poorly understood. Here we show that WRN physically interacts with the MSH2/MSH6 (MutSalpha), MSH2/MSH3 (MutSbeta) and MLH1/PMS2 (MutLalpha) heterodimers that are involved in the initiation of mismatch repair (MMR) and the rejection of homeologous recombination. MutSalpha and MutSbeta can strongly stimulate the helicase activity of WRN specifically on forked DNA structures with a 3'-single-stranded arm. The stimulatory effect of MutSalpha on WRN-mediated unwinding is enhanced by a G/T mismatch in the DNA duplex ahead of the fork. The MutLalpha protein known to bind to the MutS alpha-heteroduplex complexes has no effect on WRN-mediated DNA unwinding stimulated by MutSalpha, nor does it affect DNA unwinding by WRN alone. Our data are consistent with results of genetic experiments in yeast suggesting that MMR factors act in conjunction with a RecQ-type helicase to reject recombination between divergent sequences.

The type IIa Na(+)-dependent inorganic phosphate (Na/P(i)) cotransporter is localized in the apical membrane of proximal tubular cells and is regulated by an endocytotic pathway. Because molecular processes such as apical sorting, internalization, or subsequent degradation might be assisted by associated proteins, a yeast two-hybrid screen against the C-terminal, cytosolic tail of type IIa cotransporter was designed. Most of the potential proteins found belonged to proteins with multiple PDZ modules and were either identical/related to PDZK1 or identical to NHERF-1. Yeast trap truncation assays confined the peptide-protein association to the C-terminal amino acid residues TRL of type IIa cotransporter and to single PDZ domains of each identified protein, respectively. The specificity of these interactions were confirmed in yeast by testing other apical localized transmembraneous proteins. Moreover, the type IIa protein was recovered in vitro by glutathione S-transferase-fused PDZ proteins from isolated renal brush border membranes or from type IIa-expressing oocytes. Further, these PDZ proteins are immunohistochemically detected either in the microvilli or in the subapical compartment of proximal tubular cells. Our results suggest that the type IIa Na/P(i) cotransporter interacts with various PDZ proteins that might be responsible for the apical sorting, parathyroid hormone controlled endocytosis or the lysosomal sorting of internalized type IIa cotransporter.

Bloom's syndrome (BS) is a rare genetic disorder characterised by genomic instability and cancer susceptibility. BLM, the gene mutated in BS, encodes a member of the RecQ family of DNA helicases. Here, we identify hMLH1, which is involved in mismatch repair (MMR) and recombination, as a protein that directly interacts with BLM both in vivo and in vitro, and that the two proteins co-localise to discrete nuclear foci. The interaction between BLM and hMLH1 appears to have been evolutionarily conserved, as Sgs1p, the Saccharomyces cerevisiae homologue of BLM, interacts with yeast Mlh1p. However, cell extracts derived from BS patients show no obvious defects in MMR compared to wild-type- and BLM-complemented BS cell extracts. We conclude that the hMLH1-BLM interaction is not essential for post-replicative MMR, but, more likely, is required for some aspect of genetic recombination.

Activators of transcription are known to also play an important and direct role in activating DNA replication. However, the mechanism whereby they stimulate replication has remained elusive. One model suggests that, in the context of replication origins, transcriptional activators work by interacting with replication factors. We show that a defined, single interaction between a DNA-bound derivative of the activator Gal4 and Gal11P, a mutant form of the RNA polymerase II holoenzyme component Gal11, suffices for stimulating DNA replication as it does for transcription. Moreover, recruitment of TBP, which can activate transcription from a gene promoter, also stimulates DNA replication from an origin site. These results strongly argue that transcriptional activators may not necessarily need to contact DNA replication factors directly, but can stimulate replication by recruiting the RNA polymerase II transcription complex to DNA.

Bloom's syndrome (BS) is a genomic instability disorder characterized by cancer susceptibility. The protein defective in BS, BLM, belongs to the RecQ family of DNA helicases. In this study, we found that BLM interacts with hp150, the largest subunit of chromatin assembly factor 1 (CAF-1), in vitro and in vivo. Colocalization of a proportion of the cellular complement of these two proteins is found at specific nuclear foci coinciding with sites of DNA synthesis in the S phase. This colocalization increases in the presence of agents that damage DNA or inhibit DNA replication. In support of a functional interaction between BLM and CAF-1, we show that BLM inhibits CAF-1-mediated chromatin assembly during DNA repair in vitro. Although CAF-1 activity is not altered in BLM-deficient cells, the absence of BLM does impair the ability of CAF-1 to be mobilized within the nucleus in response to hydroxyurea treatment. Our results provide the first link between BLM and chromatin assembly coupled to DNA repair and suggest that BLM and CAF-1 function in a coordinated way to promote survival in response to DNA damage and/or replication blockade.