Perturbation of the mutated EGFR interactome identifies vulnerabilities and resistance mechanisms

We hypothesized that elucidating the interactome of epidermal growth factor receptor (EGFR) forms that are mutated in lung cancer, via global analysis of protein–protein interactions, phosphorylation, and systematically perturbing the ensuing network nodes, should offer a new, more systems‐level perspective of the molecular etiology. Here, we describe an EGFR interactome of 263 proteins and offer a 14‐protein core network critical to the viability of multiple EGFR‐mutated lung cancer cells. Cells with acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) had differential dependence of the core network proteins based on the underlying molecular mechanisms of resistance. Of the 14 proteins, 9 are shown to be specifically associated with survival of EGFR‐mutated lung cancer cell lines. This included EGFR, GRB2, MK12, SHC1, ARAF, CD11B, ARHG5, GLU2B, and CD11A. With the use of a drug network associated with the core network proteins, we identified two compounds, midostaurin and lestaurtinib, that could overcome drug resistance through direct EGFR inhibition when combined with erlotinib. Our results, enabled by interactome mapping, suggest new targets and combination therapies that could circumvent EGFR TKI resistance.     

Genetic basis of a spontaneous mutation’s expressivity

Genetic background often influences the phenotypic consequences of mutations, resulting in variable expressivity. How standing genetic variants collectively cause this phenomenon is not fully understood. Here, we comprehensively identify loci in a budding yeast cross that impact the growth of individuals carrying a spontaneous missense mutation in the nuclear-encoded mitochondrial ribosomal gene MRP20. Initial results suggested that a single large effect locus influences the mutation’s expressivity, with 1 allele causing inviability in mutants. However, further experiments revealed this simplicity was an illusion. In fact, many additional loci shape the mutation’s expressivity, collectively leading to a wide spectrum of mutational responses. These results exemplify how complex combinations of alleles can produce a diversity of qualitative and quantitative responses to the same mutation.     

PiUS (Pi uptake stimulator) is an inositol hexakisphosphate kinase

A cDNA cloned from its ability to stimulate inorganic phosphate uptake in Xenopus oocytes (phosphate uptake stimulator (PiUS)) shows significant similarity with inositol 1,4,5-trisphosphate 3-kinase. However, the expressed PiUS protein showed no detectable activity against inositol 1,4,5-trisphosphate, nor the 1,3,4,5- or 3,4,5, 6-isomers of inositol tetrakisphosphate, whereas it was very active in converting inositol hexakisphosphate (InsP(6)) to inositol heptakisphosphate (InsP(7)). PiUS is a member of a family of enzymes found in many eukaryotes and we discuss the implications of this for the functions of InsP(7) and for the evolution of inositol phosphate kinases.     

Charge-charge interactions are key determinants of the pK values of ionizable groups in ribonuclease Sa (pI=3.5) and a basic variant (pI=10.2)

The pK values of the titratable groups in ribonuclease Sa (RNase Sa) (pI=3.5), and a charge-reversed variant with five carboxyl to lysine substitutions, 5K RNase Sa (pI=10.2), have been determined by NMR at 20 degrees C in 0.1M NaCl. In RNase Sa, 18 pK values and in 5K, 11 pK values were measured. The carboxyl group of Asp33, which is buried and forms three intramolecular hydrogen bonds in RNase Sa, has the lowest pK (2.4), whereas Asp79, which is also buried but does not form hydrogen bonds, has the most elevated pK (7.4). These results highlight the importance of desolvation and charge-dipole interactions in perturbing pK values of buried groups. Alkaline titration revealed that the terminal amine of RNase Sa and all eight tyrosine residues have significantly increased pK values relative to model compounds.A primary objective in this study was to investigate the influence of charge-charge interactions on the pK values by comparing results from RNase Sa with those from the 5K variant. The solution structures of the two proteins are very similar as revealed by NMR and other spectroscopic data, with only small changes at the N terminus and in the alpha-helix. Consequently, the ionizable groups will have similar environments in the two variants and desolvation and charge-dipole interactions will have comparable effects on the pK values of both. Their pK differences, therefore, are expected to be chiefly due to the different charge-charge interactions. As anticipated from its higher net charge, all measured pK values in 5K RNase are lowered relative to wild-type RNase Sa, with the largest decrease being 2.2 pH units for Glu14. The pK differences (pK(Sa)-pK(5K)) calculated using a simple model based on Coulomb's Law and a dielectric constant of 45 agree well with the experimental values. This demonstrates that the pK differences between wild-type and 5K RNase Sa are mainly due to changes in the electrostatic interactions between the ionizable groups. pK values calculated using Coulomb's Law also showed a good correlation (R=0.83) with experimental values. The more complex model based on a finite-difference solution to the Poisson-Boltzmann equation, which considers desolvation and charge-dipole interactions in addition to charge-charge interactions, was also used to calculate pK values. Surprisingly, these values are more poorly correlated (R=0.65) with the values from experiment. Taken together, the results are evidence that charge-charge interactions are the chief perturbant of the pK values of ionizable groups on the protein surface, which is where the majority of the ionizable groups are positioned in proteins.     

Rapid identification of Arabidopsis insertion mutants by non-radioactive detection of T-DNA tagged genes

To assist in the analysis of plant gene functions we have generated a new Arabidopsis insertion mutant collection of 90 000 lines that carry the T-DNA of Agrobacterium gene fusion vector pPCV6NFHyg. Segregation analysis indicates that the average frequency of insertion sites is 1.29 per line, predicting about 116 100 independent tagged loci in the collection. The average T-DNA copy number estimated by Southern DNA hybridization is 2.4, as over 50% of the insertion loci contain tandem T-DNA copies. The collection is pooled in two arrays providing 40 PCR templates, each containing DNA from either 4000 or 5000 individual plants. A rapid and sensitive PCR technique using high-quality template DNA accelerates the identification of T-DNA tagged genes without DNA hybridization. The PCR screening is performed by agarose gel electrophoresis followed by isolation and direct sequencing of DNA fragments of amplified T-DNA insert junctions. To estimate the mutation recovery rate, 39 700 lines have been screened for T-DNA tags in 154 genes yielding 87 confirmed mutations in 73 target genes. Screening the whole collection with both T-DNA border primers requires 170 PCR reactions that are expected to detect a mutation in a gene with at least twofold redundancy and an estimated probability of 77%. Using this technique, an M2 family segregating a characterized gene mutation can be identified within 4 weeks.     

Phosphorylation of hUPF1 induces formation of mRNA surveillance complexes containing hSMG-5 and hSMG-7

Eukaryotic mRNAs containing premature termination codons (PTCs) are degraded by a process known as nonsense-mediated mRNA decay (NMD). NMD has been suggested to require the recognition of PTC by an mRNA surveillance complex containing UPF1/SMG-2. In multicellular organisms, UPF1/SMG-2 is a phosphoprotein, and its phosphorylation contributes to NMD. Here we show that phosphorylated hUPF1, the human ortholog of UPF1/SMG-2, forms a complex with human orthologs of the C. elegans NMD proteins SMG-5 and SMG-7. The complex also associates with protein phosphatase 2A (PP2A), resulting in dephosphorylation of hUPF1. Overexpression of hSMG-5 mutants that retain interaction with P-hUPF1 but which cannot induce its dephosphorylation impair NMD, suggesting that NMD requires P-hUPF1 dephosphorylation. We also show that P-hUPF1 forms distinct complexes containing different isoforms of hUPF3A. We propose that sequential phosphorylation and dephosphorylation of hUPF1 by hSMG-1 and PP2A, respectively, contribute to the remodeling of the mRNA surveillance complex.     

Isolation of moderately infectious Borrelia burgdorferi sensu stricto from attenuated cultures by using complement-mediated,antibody-dependent lysis selection technique in a mammalian tissue co-culture system

We investigated the association between complement resistance and phenotypes of pathogenicity of Borrelia burgdorferi sensu lato isolates cultivated in a LEW/N rat tibiotarsal joint-derived tissue feeder layer-supported co-culture system. Guinea pig complement and immune serum raised in LHS/Ss hamsters caused complete lysis of B. burgdorferi sensu stricto isolate 297, B. afzelii and B. garinii in Barbour-Stoenner-Kelly's medium; however, tissue co-cultured B. burgdorferi sensu stricto contained complement escape variants. The arthritogenicity and infectivity of these variants were tested in 3-week-old Syrian hamsters and in a vaccinated hamster model in which formalin-killed B. burgdorferi sensu stricto C-1-11 vaccinated animals develop severe arthritis after challenge with live, pathogenic, low-passage 297 isolate. Non-animal-passaged complement escape variants were infectious in both animal models as demonstrated by re-isolation from the infected animals and competitive PCR. IP injection of animal-passaged complement escape variants caused development of severe arthritis in vaccinated animals 5 weeks post-injection; animal passage of complement escape variants was necessary for isolation of arthritogenic spirochetes from high-passaged, non-arthritogenic, attenuated borrelia cultures. Complement escape variants synthesized outer surface protein E as demonstrated by SDS-PAGE and western blotting analyses. The complement-mediated selection technique in tissue co-culture provides a novel approach to the studies of Lyme disease, enables us to isolate pathogenically distinct borrelia populations from attenuated cultures and prepare a moderately infectious, non-pathogenic live vaccine against this illness.