B‐type natriuretic peptide and extracellular matrix protein interactions in human cardiac fibroblasts

Cardiac fibroblasts (CFs) regulate myocardial remodeling by proliferating, differentiating, and secreting extracellular matrix (ECM) proteins. B‐type natriuretic peptide (BNP) is anti‐fibrotic, inhibits collagen production, augments matrix metalloproteinases, and suppresses CF proliferation. Recently, we demonstrated that the ECM protein fibronectin (FN) augmented production of BNP's second messenger, 3′, 5′ cyclic guanosine monophosphate (cGMP) in CFs, supporting crosstalk between FN, BNP, and its receptor, natriuretic peptide receptor A (NPR‐A). Here, we address the specificity of FN to augment cGMP generation by investigating other matrix proteins, including collagen IV which contains RGD motifs and collagen I and poly‐L ‐lysine, which have no RGD domain. Collagen IV showed increased cGMP generation to BNP similar to FN. Collagen I and poly‐L ‐lysine had no effect. As FN also interacts with integrins, we then examined the effect of integrin receptor antibody blockade on BNP‐mediated cGMP production. On FN plates, antibodies blocking RGD‐binding domains of several integrin subtypes had little effect, while a non‐RGD domain interfering integrin αvβ3 antibody augmented cGMP production. Further, on uncoated plates, integrin αvβ3 blockade continued to potentiate the BNP/cGMP response. These studies suggest that both RGD containing ECM proteins and integrins may interact with BNP/NPR‐A to modulate cGMP generation. J. Cell. Physiol. 225: 251–255, 2010. © 2010 Wiley‐Liss, Inc.     

Specific copper transfer from the Cox17 metallochaperone to both Sco1 and Cox11 in the assembly of yeast cytochrome C oxidase

The assembly of the copper sites in cytochrome c oxidase involves a series of accessory proteins, including Cox11, Cox17, and Sco1. The two mitochondrial inner membrane proteins Cox11 and Sco1 are thought to be copper donors to the Cu(B) and Cu(A) sites of cytochrome oxidase, respectively, whereas Cox17 is believed to be the copper donor to Sco1 within the intermembrane space. In this report we show Cox17 is a specific copper donor to both Sco1 and Cox11. Using in vitro studies with purified proteins, we demonstrate direct copper transfer from CuCox17 to Sco1 or Cox11. The transfer is specific because no transfer occurs to heterologous proteins, including bovine serum albumin and carbonic anhydrase. In addition, a C57Y mutant of Cox17 fails to transfer copper to Sco1 but is competent for copper transfer to Cox11. The in vitro transfer studies were corroborated by a yeast cytoplasm expression system. Soluble domains of Sco1 and Cox11, lacking the mitochondrial targeting sequence and transmembrane domains, were expressed in the yeast cytoplasm. Metallation of these domains was strictly dependent on the co-expression of Cox17. Thus, Cox17 represents a novel copper chaperone that delivers copper to two proteins.     

Rapid cooperative two-state folding of a miniature alpha-beta protein and design of a thermostable variant

There is a great deal of interest in developing small stably folded miniature proteins. A limited number of these molecules have been described, however they typically have not been characterized in depth. In particular, almost no detailed studies of the thermodynamics and folding kinetics of these proteins have been reported. Here we describe detailed studies of the thermodynamics and kinetics of folding of a 39 residue mixed alpha-beta protein (NTL9(1-39)) derived from the N-terminal domain of the ribosomal protein L9. The protein folds cooperatively and rapidly in a two-state fashion to a native state typical of those found for normal globular proteins. At pH 5.4 in 20mM sodium acetate, 100mM NaCl the temperature of maximum stability is 6 degrees C, the t(m) is 65.3 degrees C, deltaH degrees (t(m)) is between 24.6 kcalmol(-1) and 26.3 kcalmol(-1), and deltaC(p) degrees is 0.38 kcalmol(-1)deg(-1). The thermodynamic parameters are in the range expected on the basis of per residue values determined from databases of globular proteins. H/2H exchange measurements reveal a set of amides that exchange via global unfolding, exactly as expected for a normal cooperatively folded globular protein. Kinetic measurements show that folding is two-state folding. The folding rate is 640 s(-1) and the value of deltaG degrees calculated from the folding and unfolding rates is in excellent agreement with the equilibrium value. A designed thermostable variant, generated by mutating K12 to M, was characterized and found to have a t(m) of 82 degrees C. Equilibrium and kinetic measurements demonstrate that its folding is cooperative and two-state.     

Community trust of government and non-governmental organizations during the 2014-16 Ebola epidemic in Liberia

The West African Ebola Virus Disease epidemic of 2014-16 cost more than 11,000 lives. Interventions targeting key behaviors to curb transmission, such as safe funeral practices and reporting and isolating the ill, were initially unsuccessful in a climate of fear, mistrust, and denial. Building trust was eventually recognized as essential to epidemic response and prioritized, and trust was seen to improve toward the end of the epidemic as incidence fell. However, little is understood about how and why trust changed during Ebola, what factors were most influential to community trust, and how different institutions might have been perceived under different levels of exposure to the outbreak. In this large-N household survey conducted in Liberia in 2018, we measured self-reported trust over time retrospectively in three different communities with different exposures to Ebola. We found trust was consistently higher for non-governmental organizations than for the government of Liberia across all time periods. Trust reportedly decreased significantly from the start to the peak of the epidemic in the study site of highest Ebola incidence. This finding, in combination with a negative association found between knowing someone infected and trust of both iNGOs and the government, indicates the experience of Ebola may have itself caused a decline of trust in the community. These results suggest that national governments should aim to establish trust when engaging communities to change behavior during epidemics. Further research on the relationship between trust and epidemics may serve to improve epidemic response efficacy and behavior uptake.     

Beyond fecundity and longevity: trade-offs between reproduction and survival mediated by behavioural responses of the seed beetle, Callosobruchus maculatus

Abstract.  Many studies of life-history traits have failed to find trade-offs where they are predicted by theory. A hypothesis that explains the lack of trade-offs between fecundity and longevity in the seed beetle, Callosobruchus maculatus , is proposed. By manipulating host availability time and host size, trade-offs mediated by behavioural responses of the female to adapt to environmental change are tested. Females show no decrease in lifetime fecundity when host availability time is limited to only 4 h on each day. However, longevity significantly increases when the female is provided with small beans after host deprivation. Because neither acquisition, nor utilization by females of these four manipulation treatments significantly differs, studies are carried out to demonstrate whether the energy shifted from increased longevity without decreasing fecundity. Providing abundant small or large beans each day directly after host deprivation, significantly increases the number of daily eggs laid by the female for several days, whereas the female decreases the uniformity of her egg dispersion only when small beans are provided. Therefore, the female shows a response to a change in the environment by adjusting egg-laying rate and/or egg-dispersion pattern. This may change the traits of reproduction and survival. Because energy allocations can be shifted between components of reproduction (e.g. host-selection behaviour and fecundity) or between reproduction and survival, fecundity and longevity may be inappropriate indices for trade-off analyses in this study. A framework for exploring the costs of reproduction mediated by physiological and behavioural changes in C. maculatus is proposed and discussed.     

Human Sco1 and Sco2 function as copper-binding proteins

The function of human Sco1 and Sco2 is shown to be dependent on copper ion binding. Expression of soluble domains of human Sco1 and Sco2 either in bacteria or the yeast cytoplasm resulted in the recovery of copper-containing proteins. The metallation of human Sco1, but not Sco2, when expressed in the yeast cytoplasm is dependent on the co-expression of human Cox17. Two conserved cysteines and a histidyl residue, known to be important for both copper binding and in vivo function in yeast Sco1, are also critical for in vivo function of human Sco1 and Sco2. Human and yeast Sco proteins can bind either a single Cu(I) or Cu(II) ion. The Cu(II) site yields S-Cu(II) charge transfer transitions that are not bleached by weak reductants or chelators. The Cu(I) site exhibits trigonal geometry, whereas the Cu(II) site resembles a type II Cu(II) site with a higher coordination number. To identify additional potential ligands for the Cu(II) site, a series of mutant proteins with substitutions in conserved residues in the vicinity of the Cu(I) site were examined. Mutation of several conserved carboxylates did not alter either in vivo function or the presence of the Cu(II) chromophore. In contrast, replacement of Asp238 in human or yeast Sco1 abrogated the Cu(II) visible transitions and in yeast Sco1 attenuated Cu(II), but not Cu(I), binding. Both the mutant yeast and human proteins were nonfunctional, suggesting the importance of this aspartate for normal function. Taken together, these data suggest that both Cu(I) and Cu(II) binding are critical for normal Sco function.     

A general model for preferential hetero-oligomerization of LIN-2/7 domains: mechanism underlying directed assembly of supramolecular signaling complexes

LIN-2/7 (L27) domains are protein interaction modules that preferentially hetero-oligomerize, a property critical for their function in directing specific assembly of supramolecular signaling complexes at synapses and other polarized cell-cell junctions. We have solved the solution structure of the heterodimer composed of the L27 domains from LIN-2 and LIN-7. Comparison of this structure with other L27 domain structures has allowed us to formulate a general model for why most L27 domains form an obligate heterodimer complex. L27 domains can be divided in two types (A and B), with each heterodimer comprising an A/B pair. We have identified two keystone positions that play a central role in discrimination. The residues at these positions are energetically acceptable in the context of an A/B heterodimer, but would lead to packing defects or electrostatic repulsion in the context of A/A and B/B homodimers. As predicted by the model, mutations of keystone residues stabilize normally strongly disfavored homodimers. Thus, L27 domains are specifically optimized to avoid homodimeric interactions.