Quantitative GTPase affinity purification identifies Rho family protein interaction partners

Although Rho GTPases are essential molecular switches involved in many cellular processes, an unbiased experimental comparison of their interaction partners was not yet performed. Here, we develop quantitative GTPase affinity purification (qGAP) to systematically identify interaction partners of six Rho GTPases (Cdc42, Rac1, RhoA, RhoB, RhoC, RhoD) depending on their nucleotide loading state. The method works with cell line or tissue-derived protein lysates in combination with SILAC-based or label free quantification, respectively. We demonstrate that qGAP identifies known and novel binding partners that can be validated in an independent assay. Our interaction network for six Rho GTPases contains many novel binding partners, reveals highly promiscuous interaction of several effectors and mirrors evolutionary relationships among Rho GTPases

Observed Limits on Charge Exchange Contributions to the Diffuse X-ray Background

We present a high resolution spectrum of the diffuse X-ray background from 0.1 to 1 keV for a ~1 region of the sky centered at l=90, b=+60 using a 36-pixel array of microcalorimeters flown on a sounding rocket. With an energy resolution of 11 eV FWHM below 1 keV, the spectrum's observed line ratios help separate charge exchange contributions originating within the heliosphere from thermal emission of hot gas in the interstellar medium. The X-ray sensitivity below 1 keV was reduced by about a factor of four from contamination that occurred early in the flight, limiting the significance of the results. The observed centroid of helium-like O VII is 568+2-3 eV at 90% confidence. Since the centroid expected for thermal emission is 568.4 eV while for charge exchange is 564.2 eV, thermal emission appears to dominate for this line complex, consistent with much of the high-latitude O VII emission originating in 2-3 x 10^6 K gas in the Galactic halo. On the other hand, the observed ratio of C VI Ly gamma to Ly alpha is 0.3+-0.2. The expected ratios are 0.04 for thermal emission and 0.24 for charge exchange, indicating that charge exchange must contribute strongly to this line and therefore potentially to the rest of the ROSAT R12 band usually associated with 10^6 K emission from the Local Hot Bubble. The limited statistics of this experiment and systematic uncertainties due to the contamination require only >32% thermal emission for O VII and >20% from charge exchange for C VI at the 90% confidence level. An experimental gold coating on the silicon substrate of the array greatly reduced extraneous signals induced on nearby pixels from cosmic rays passing through the substrate, reducing the triggered event rate by a factor of 15 from a previous flight of the instrument.Comment: 14 pages, 7 figures, to be published in Ap

A new role of the Rac-GAP β2-chimaerin in cell adhesion reveals opposite functions in breast cancer initiation and tumor progression

β2-chimaerin is a Rac1-specific negative regulator and a candidate tumor suppressor in breast cancer but its precise function in mammary tumorigenesis in vivo is unknown. Here, we study for the first time the role of β2-chimaerin in breast cancer using a mouse model and describe an unforeseen role for this protein in epithelial cell-cell adhesion. We demonstrate that expression of β2-chimaerin in breast cancer epithelial cells reduces E-cadherin protein levels, thus loosening cell-cell contacts. In vivo, genetic ablation of β2-chimaerin in the MMTV-Neu/ErbB2 mice accelerates tumor onset, but delays tumor progression. Finally, analysis of clinical databases revealed an inverse correlation between β2-chimaerin and E-cadherin gene expressions in Her2+ breast tumors. Furthermore, breast cancer patients with low β2-chimaerin expression have reduced relapse free survival but develop metastasis at similar times. Overall, our data redefine the role of β2-chimaerin as tumor suppressor and provide the first in vivo evidence of a dual function in breast cancer, suppressing tumor initiation but favoring tumor progression

Interaction modulation through arrays of clustered methyl-arginine protein modifications

Systematic analysis of human arginine methylation identifies two distinct signaling modes;either isolated modifications akin to canonical post-translational modification regulation, or clustered arrays within disordered protein sequence. Hundreds of proteins contain these methyl-arginine arrays and are more prone to accumulate mutations and more tightly expression-regulated than dispersed methylation targets. Arginines within an array in the highly methylated RNA-binding protein synaptotagmin binding cytoplasmic RNA interacting protein (SYNCRIP) were experimentally shown to function in concert, providing a tunable protein interaction interface. Quantitative immunoprecipitation assays defined two distinct cumulative binding mechanisms operating across 18 proximal arginine-glycine (RG) motifs in SYNCRIP. Functional binding to the methyltransferase PRMT1 was promoted by continual arginine stretches, whereas interaction with the methyl-binding protein SMN1 was arginine content-dependent irrespective of linear position within the unstructured region. This study highlights how highly repetitive modifiable amino acid arrays in low structural complexity regions can provide regulatory platforms, with SYNCRIP as an extreme example how arginine methylation leverages these disordered sequences to mediate cellular interactions

A characteristic lengthscale causes anomalous size effects and boundary programmability in mechanical metamaterials

The architecture of mechanical metamaterialsis designed to harness geometry, non-linearity and topology to obtain advanced functionalities such as shape morphing, programmability and one-way propagation. While a purely geometric framework successfully captures the physics of small systems under idealized conditions, large systems or heterogeneous driving conditions remain essentially unexplored. Here we uncover strong anomalies in the mechanics of a broad class of metamaterials, such as auxetics, shape-changers or topological insulators: a non-monotonic variation of their stiffness with system size, and the ability of textured boundaries to completely alter their properties. These striking features stem from the competition between rotation-based deformations---relevant for small systems---and ordinary elasticity, and are controlled by a characteristic length scale which is entirely tunable by the architectural details. Our study provides new vistas for designing, controlling and programming the mechanics of metamaterials in the thermodynamic limit.Comment: Main text has 4 pages, 4 figures + Methods and Supplementary Informatio

The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) family

The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) enzymes are secreted, multi-domain matrix-associated zinc metalloendopeptidases that have diverse roles in tissue morphogenesis and patho-physiological remodeling, in inflammation and in vascular biology. The human family includes 19 members that can be sub-grouped on the basis of their known substrates, namely the aggrecanases or proteoglycanases (ADAMTS1, 4, 5, 8, 9, 15 and 20), the procollagen N-propeptidases (ADAMTS2, 3 and 14), the cartilage oligomeric matrix protein-cleaving enzymes (ADAMTS7 and 12), the von-Willebrand Factor proteinase (ADAMTS13) and a group of orphan enzymes (ADAMTS6, 10, 16, 17, 18 and 19). Control of the structure and function of the extracellular matrix (ECM) is a central theme of the biology of the ADAMTS, as exemplified by the actions of the procollagen-N-propeptidases in collagen fibril assembly and of the aggrecanases in the cleavage or modification of ECM proteoglycans. Defects in certain family members give rise to inherited genetic disorders, while the aberrant expression or function of others is associated with arthritis, cancer and cardiovascular disease. In particular, ADAMTS4 and 5 have emerged as therapeutic targets in arthritis. Multiple ADAMTSs from different sub-groupings exert either positive or negative effects on tumorigenesis and metastasis, with both metalloproteinase-dependent and -independent actions known to occur. The basic ADAMTS structure comprises a metalloproteinase catalytic domain and a carboxy-terminal ancillary domain, the latter determining substrate specificity and the localization of the protease and its interaction partners; ancillary domains probably also have independent biological functions. Focusing primarily on the aggrecanases and proteoglycanases, this review provides a perspective on the evolution of the ADAMTS family, their links with developmental and disease mechanisms, and key questions for the future

Alignment of a digital watershed and land use game to national education standards

Digital games, especially simulations, have supported student learning outcomes in the areas of science and agriculture in classrooms and nonformal settings. Simulations contribute robustly to student achievement in science, technology, engineering, and mathematics (STEM), and agriculture content areas, especially when they are aligned with national education standards. The People in Ecosystems Watershed Integration (PEWI) simulation is a digital game that was evaluated for fit to two national standards: the Next Generation Science Standards (NGSS) and the Agriculture, Food, and Natural Resources Standards (AFNR). The evaluation of alignment of PEWI to NGSS provided “extensive” evidence on a four‐point scale for meeting Criterion A: Explaining phenomenon/designing solutions; Criterion B: 3‐D learning, science and engineering practices, rated for three areas: (a) “extensive” for science and engineering practices, (b) “adequate” for disciplinary core ideas, and (c) “extensive” for cross‐cutting concepts. Additionally, PEWI aligned with nine high school–level NGSS student performance expectations categories. For AFNR Standards, the PEWI evaluation provided evidence for alignment to 10 standards and 17 indicators from the AFNR areas of Environmental Service Systems, Natural Resource Systems, and Plant Systems

Identification of Giardia lamblia DHHC Proteins and the Role of Protein S-palmitoylation in the Encystation Process

Protein S-palmitoylation, a hydrophobic post-translational modification, is performed by protein acyltransferases that have a common DHHC Cys-rich domain (DHHC proteins), and provides a regulatory switch for protein membrane association. In this work, we analyzed the presence of DHHC proteins in the protozoa parasite Giardia lamblia and the function of the reversible S-palmitoylation of proteins during parasite differentiation into cyst. Two specific events were observed: encysting cells displayed a larger amount of palmitoylated proteins, and parasites treated with palmitoylation inhibitors produced a reduced number of mature cysts. With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome. These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades. Although all Giardia DHHC proteins were found to be present in trophozoites and encysting cells, these proteins showed a different intracellular localization in trophozoites and seemed to be differently involved in the encystation process when they were overexpressed. dhhc transgenic parasites showed a different pattern of cyst wall protein expression and yielded different amounts of mature cysts when they were induced to encyst. Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation.Fil: Merino, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Zamponi, Nahuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Vranych, Cecilia Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Touz, Maria Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Ropolo, Andrea Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentin