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ABSTRACTCancer is the second leading cause of death globally, and colorectal cancer (CRC) is among the five most common cancers. The small GTPase KRAS is an oncogene that is mutated in ~30% of all CRCs. Pharmacological treatments of CRC are currently unsatisfactory, but much hope rests on network-centric approaches to drug development and cancer treatment. These approaches, however, require a better understanding of how networks downstream of Ras oncoproteins are connected in a particular tissue context – here colon and CRC. Previously we have shown that competition for binding to a ‘hub’ protein, such as Ras, can induce a rewiring of signal transduction networks. In this study, we analysed 56 established and predicted effectors that contain a structural domain with the potential ability to bind to Ras oncoproteins and their link to pathways coordinating intestinal homoeostasis and barrier function. Using protein concentrations in colon tissue and Ras-effector binding affinities, a computational network model was generated that predicted how effectors differentially and competitively bind to Ras in colon context. The model also predicted both qualitative and quantitative changes in Ras-effector complex formations with increased levels of active Ras – to simulate its upregulation in cancer – simply as an emergent property of competition for the same binding interface on the surface of Ras. We also considered how the number of Ras-effector complexes at the membrane can be increased by additional domains present in some effectors that are recruited to the membrane in response to specific conditions (inputs/stimuli/growth factors) in colon context and CRC.

IMPORTANCE: Age-related macular degeneration (AMD) is a common threat to vision loss in individuals older than 50 years. While neovascular complications in AMD are treatable, there is currently no therapy for geographic atrophy secondary to AMD. Geographic atrophy lesion progression over time shows considerable interindividual variability, but little is known about prognostic factors. OBJECTIVE: To elucidate the contribution of common genetic variants to geographic atrophy lesion growth. DESIGN, SETTING, AND PARTICIPANTS: This pooled analysis combined 4 independent studies: the Fundus Autofluorescence Imaging in Age-Related Macular Degeneration (FAM) study, the Directional Spread in Geographic Atrophy (DSGA) study, the Age-Related Eye Disease Study (AREDS), and the Geographic Atrophy Treatment Evaluation (GATE) study. Each provided data for geographic atrophy lesion growth in specific designs. Patients with geographic atrophy secondary to AMD were recruited to these studies. Genotypes were retrieved through the database of Genotypes and Phenotypes (for AREDS) or generated at the Cologne Center for Genomics (for FAM, DSGA, and GATE). MAIN OUTCOMES: The correlation between square root–transformed geographic atrophy growth rate and 7 596 219 genetic variants passing quality control was estimated using linear regression. The calculations were adjusted for known factors influencing geographic atrophy growth, such as the presence of bilateral geographic atrophy as well as the number of lesion spots and follow-up times. MAIN OUTCOMES AND MEASURES: Slopes per allele, 95% CIs, and P values of genetic variants correlated with geographic atrophy lesion growth. RESULTS: A total of 935 patients (mean [SD] age, 74.7 [7.8] years; 547 female participants [59.0%]) were included. Two gene loci with conservative genome-wide significance were identified. Each minor allele of the genome-wide associated variants increased the geographic atrophy growth rate by a mean of about 15% or 0.05 mm per year. Gene prioritization within each locus suggests the protein arginine methyltransferase 6 gene (PRMT6; chromosome 1; slope, 0.046 [95% CI, 0.026-0.066]; P = 4.09 × 10−8) and the lanosterol synthase gene (LSS; chromosome 21; slope, 0.105 [95% CI, 0.068-0.143]; P = 4.07 × 10−7) as the most likely progression-associated genes. CONCLUSIONS AND RELEVANCE: These data provide further insight into the genetic architecture of geographic atrophy lesion growth. Geographic atrophy is a clinical outcome with a high medical need for effective therapy. The genes PRMT6 and LSS are promising candidates for future studies aimed at understanding functional aspects of geographic atrophy progression and also for designing novel and targeted treatment options.

Age-related macular degeneration (AMD) is the leading cause of blindness in industrialised countries, and thereby a major individual but also a socio-economic burden. Y chromosome loss in nucleated blood cells has been implicated in age-related diseases such as Alzheimer disease and was shown to be caused by increasing age, smoking and genetic factors. Mosaic loss of Y chromosome (mLOY) in peripheral blood was estimated from normalised dosages of genotyping chip data covering the male-specific region of the Y chromosome. After quality control, we assessed the association of mLOY on AMD risk in 5772 male cases and 6732 male controls. In controls the prevalence of mLOY increased significantly with age, which is consistent with previous reports. Importantly, mLOY was associated with late-stage AMD with genome-wide significance (OR: 1.332 [95% CI: 1.206; 1.472], P= 1.60e-08), independent of age, the AMD genetic risk score and the first two principle components of ancestry. Additionally conditioning on smoking behaviour had no influence on the observed association strength. mLOY was strongest associated in individuals aged between 65 and 75 years. Taken together, mLOY is significantly associated with risk for AMD, independent of known and potential confounding factors.

The understanding of interaction dynamics in signaling pathways can shed light on pathway architecture and provide insights into targets for intervention. Here, we explored the relevance of kinetic rate constants of a key upstream osmosensor in the yeast high-osmolarity glycerol-mitogen-activated protein kinase (HOG-MAPK) pathway to signaling output responses. We created mutant pairs of the Sln1-Ypd1 complex interface that caused major compensating changes in the association (kon) and dissociation (koff) rate constants (kinetic perturbations) but only moderate changes in the overall complex affinity (Kd). Yeast cells carrying a Sln1-Ypd1 mutant pair with moderate increases in konand koffdisplayed a lower threshold of HOG pathway activation than wild-type cells. Mutants with higher konand koffrates gave rise to higher basal signaling and gene expression but impaired osmoadaptation. Thus, the konand koffrates of the components in the Sln1 osmosensor determine proper signaling dynamics and osmoadaptation.

Relating protein concentration to cell-type-specific responses is one of the remaining challenges for obtaining a quantitative systems level understanding of mammalian signaling. Here we used mass-spectrometry (MS)- and antibody-based quantitative proteomic approaches to measure protein abundances for 75% of a hand-curated reconstructed ErbB network of 198 proteins, in two established cell types (HEK293 and MCF-7) and in primary keratinocyte cells. Comparison with other quantitative studies allowed building a set of ErbB network proteins expressed in all cells and another which are cell-specific and could impart specific properties to the network. As a proof-of-concept of the importance of protein concentration, we generated a small simplified mathematical model encompassing ligand binding, followed by receptor dimerization, activation, and degradation. The model predicts ErbB phosphorylation in HEK293, MCF-7, and keratinocyte cells simply by incorporating cell-type-specific ErbB1, ErbB2, and caveolin-1 abundances but otherwise contains similar rate constants. Altogether, the data provide a resource for protein abundances and localization to be included in larger mathematical models, enabling the generation of cell-type-specific computational models. MS data have been deposited to the ProteomeXchange via PRIDE (with identifier PXD000623) and PASSEL (with identifier PASS00372).

Transnational advocacy networks (TANs) are receiving increased attention in the international relations literature. One central interest has addressed the efforts of local activists to circumvent hostile domestic governments by appealing abroad (the ‘boomerang hypothesis’). When success in the domestic policy process is denied to civil society actors, they reach out to international partners who in turn pressure the local government. In the parlance of the policy making literature, they ‘venue shop’. These outsiders may be foreign governments, intergovernmental organizations or international non-governmental organizations. Previous studies indicate that international actors can be powerful advocates for local causes who make success of the TAN more likely. This article assesses the boomerang hypothesis by focussing on the network that arose in response to the near-extermination of black-necked swans in the Carlos Anwandter Natural Sanctuary (Chile) after the opening of a pulp mill. Specifically, it explores the complicated relationship of local ‘boomerang throwers’ and international ‘boomerang recipients’. We argue that securing international participants does not guarantee TAN success. In particular, we find that involvement of governmental actors in a TAN lend power to the cause, but at the same time may dilute the mission of the TAN, leading to less-than-optimal outcomes from the perspective of the local campaign initiators. Additionally, we consider how the content of network interactions may influence its structure, which in turn affects the outcome of transnational campaigns. Finally, our research points to the difficulty of assessing success. In the present case, many local Chilean activists were disappointed that the pulp mill they held responsible for environmental damages was not shut down. At the same time, the company in question has become more responsive to demands of environmental sustainability, and the campaign raised environmental awareness in Chile. This exploratory research has important implications for academic research of TANs, as well as for practitioners working on transnational campaigns. Our data suggest caution in employing the ‘boomerang’ mechanism, especially when the international actors are governments. What returns with the boomerang may prove unsatisfying to those throwing it. Relatedly, we find that potential TAN participants must weigh existing relationships with TAN targets against the benefits of joining the TAN. More generally, these findings support the constraints on venue shopping that arise from limited information and venue stickiness. Taken together, these findings suggest a caution for those examining the density of network interactions to measure strength. The content of interactions in a network may be as important as their frequency.

Ras proteins belong to the superfamily of small GTP-binding proteins which have the ability to recognize and bind to several sets of effector proteins, thereby communicating signals into different pathways. Although the Ras proteins have almost identical amino acid residues in the effector binding region, and the Ras binding domains have a similar ubiquitin-like topology, the affinity constants span three orders of magnitude. Moreover, large differences in the individual enthalpic and entropic contributions are observed. Another important feature of Ras-effector interactions is the charge complementarity found between the proteins in the complex. As a result, the association rate constant is very high and contributes significantly to the affinity of the complex. Differences in binding affinities are mainly due to changes in the association rate constants with the dissociation rate constants being at a similar range. Here, we discuss the importance of understanding protein complex formation in signal transduction pathways on a molecular level, both, thermodynamically and kinetically. Although the focus is mainly on Ras-effector interactions, comparisons of different binding modes, thermodynamics and electrostatics of various effectors in complex with members of the Rho, Rab, Arf, and Ran families will also be considered. This information could be important to understand the specificity in different pathways and for a rational design of compounds which block specific pathways.

Small GTPases of the Ras family are major players of signal transduction in eukaryotic cells. They receive signals from a number of receptors and transmit them to a variety of effectors. The distribution of signals to different effector molecules allows for the generation of opposing effects like proliferation and differentiation. To understand the specificity of Ras signaling, we investigated the activation of RalGDS, one of the Ras effector proteins with guanine-nucleotide exchange factor activity for Ral. We determined the GTP level on RalA and showed that the highly conserved Ras binding domain (RBD) of RalGDS, which mediates association with Ras, is important but not sufficient to explain the stimulation of the exchange factor. Although a point mutation in the RBD of RalGDS, which abrogates binding to Ras, renders RalGDS independent to activated Ras, an artificially membrane-targeted version of RalGDS lacking its RBD could still be activated by Ras. The switch II region of Ras is involved in the activation, because the mutant Y64W in this region is impaired in the RalGDS activation. Furthermore, it is shown that Rap1, which was originally identified as a Ras antagonist, can block Ras-mediated RalGDS signaling only when RalGDS contains an intact RBD. In addition, kinetic studies of the complex formation between RalGDS-RBD and Ras suggest that the fast association between RalGDS and Ras, which is analogous to the Ras/Raf case, achieves signaling specificity. Conversely, the Ras·RalGDS complex has a short lifetime of 0.1 s and Rap1 forms a long-lived complex with RalGDS, possibly explaining its antagonistic effect on Ras.

Competition among mutually exclusive binding partners contributes to context-dependent differences in signaling responses.

Ras–c-Raf association rates affect downstream signaling in the absence of negative feedback but not its presence.