von Willebrand factor (VWF) propeptide binding to VWF D'D3 domain attenuates platelet activation and adhesion.

Noncovalent association between the von Willebrand factor (VWF) propeptide (VWFpp) and mature VWF aids N-terminal multimerization and protein compartmentalization in storage granules. This association is currently thought to dissipate after secretion into blood. In the present study, we examined this proposition by quantifying the affinity and kinetics of VWFpp binding to mature VWF using surface plasmon resonance and by developing novel anti-VWF D'D3 mAbs. Our results show that the only binding site for VWFpp in mature VWF is in its D'D3 domain. At pH 6.2 and 10mM Ca(2+), conditions mimicking intracellular compartments, VWFpp-VWF binding occurs with high affinity (K(D) = 0.2nM, k(off) = 8 × 10(-5) s(-1)). Significant, albeit weaker, binding (K(D) = 25nM, k(off) = 4 × 10(-3) s(-1)) occurs under physiologic conditions of pH 7.4 and 2.5mM Ca(2+). This interaction was also observed in human plasma (K(D) = 50nM). The addition of recombinant VWFpp in both flow-chamber-based platelet adhesion assays and viscometer-based shear-induced platelet aggregation and activation studies reduced platelet adhesion and activation partially. Anti-D'D3 mAb DD3.1, which blocks VWFpp binding to VWF-D'D3, also abrogated platelet adhesion, as shown by shear-induced platelet aggregation and activation studies. Our data demonstrate that VWFpp binding to mature VWF occurs in the circulation, which can regulate the hemostatic potential of VWF by reducing VWF binding to platelet GpIbα.

SWAT model applications: From hydrological processes to ecosystem services.

Ecosystem services in bolstering human well-being and steering environmental management garnered increasing recognition. In this realm, the Soil and Water Assessment Tool (SWAT) rose as an instrumental tool in ecosystem services. The heterogeneous applications of SWAT across diverse studies underscore an imperative for bibliometric analysis to decipher these evolving trends. This study endeavors to execute a comprehensive analysis of SWAT's application for ecosystem services, delineating key thematic development and exploring its utilization in ecosystem services. We conducted a comprehensive literature review by searching the Web of Science database, retrieving a total of 534 articles. The CiteSpace facilitated our co-citation analysis, enabling the identification of seminal publications and burgeoning themes within SWAT. Our analysis delineated thematic development in SWAT pertaining to ecosystem services. Initially concentrated on hydrological processes, the focus progressively broadened to encompass comprehensive ecosystem services evaluations. We examined 81 peer-reviewed publications directly related to ecosystem services, and most of them addressed certain ecosystem services, such as water yield, soil retention, regulation of water flow, food, and carbon storage. SWAT holds a unique advantage in quantifying water-related processes. Future studies should focus more on ecosystem service flows based on SWAT, which contributes to elucidating the relationship between nature and humans, facilitating comprehensive ecosystem management.

Different electrostatic potentials define ETGE and DLG motifs as hinge and latch in oxidative stress response.

Nrf2 is the regulator of the oxidative/electrophilic stress response. Its turnover is maintained by Keap1-mediated proteasomal degradation via a two-site substrate recognition mechanism in which two Nrf2-Keap1 binding sites form a hinge and latch. The E3 ligase adaptor Keap1 recognizes Nrf2 through its conserved ETGE and DLG motifs. In this study, we examined how the ETGE and DLG motifs bind to Keap1 in a very similar fashion but with different binding affinities by comparing the crystal complex of a Keap1-DC domain-DLG peptide with that of a Keap1-DC domain-ETGE peptide. We found that these two motifs interact with the same basic surface of either Keap1-DC domain of the Keap1 homodimer. The DLG motif works to correctly position the lysines within the Nrf2 Neh2 domain for efficient ubiquitination. Together with the results from calorimetric and functional studies, we conclude that different electrostatic potentials primarily define the ETGE and DLG motifs as a hinge and latch that senses the oxidative/electrophilic stress.

Application of fluorescence spectroscopy to quantify shear-induced protein conformation change.

Rapid and robust methods are required to quantify the effect of hydrodynamic shear on protein conformation change. We evaluated such strategies in this work and found that the binding of the fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to hydrophobic pockets in the blood protein von Willebrand factor (VWF) is enhanced upon the application of fluid shear to the isolated protein. Significant structural changes were observed when the protein was sheared at shear rates >or= 6000/s for approximately 3.5 min. The binding of bis-ANS to multimeric VWF, but not dimeric VWF or control protein bovine serum albumin, was enhanced upon fluid shear application. Thus, high-molecular-weight VWF is more susceptible to conformation change upon tensile loading. Although bis-ANS itself did not alter the conformation of VWF, it stabilized protein conformation once it bound the sheared molecule. Bis-ANS binding to VWF was reduced when the sheared protein was allowed to relax before dye addition. Taken together with functional data in the literature, our results suggest that shear-induced conformation changes in VWF reported by bis-ANS correlate well with the normal function of the protein under physiological/pathological fluid flow conditions. Further, this study introduces the fluorescent dye bis-ANS as a tool that may be useful in studies of shear-induced protein conformation change.

The role of glycosylation in the function of a 48-kDa glycoprotein from carrot.

Carrot extracellular dermal glycoprotein (EDGP) may play an important role in plant defense systems and in signal transduction. Our experiments show that differences in pI values of EDGP isoforms are caused by differences in amino acid sequence and not by heterogeneity in phosphorylation. The binding affinity of native EDGP for a 4-kDa hormone-like peptide from soybean was approximately one-third that of deglycosylated EDGP, and deglycosylation of EDGP caused complete loss of its ability to inhibit xyloglucan-specific endo-beta-1,4-glucanase. Experiments using tunicamycin-treated carrot cell cultures showed that glycosylation is essential for correct EDGP folding and secretion, and that tunicamycin does not affect EDGP gene transcription.

Mass spectrometric analysis of posttranslational modifications of a carrot extracellular glycoprotein.

Expression of extracellular dermal glycoprotein (EDGP) is induced by biotic or abiotic stress. The amino acid sequence alignment showed that EDGP shared significant homology with proteins from legumes, tomato, Arabidopsis, wheat, and cotton. These proteins are involved in signal transduction or stress response systems. Most of the Cys residues in these proteins are conserved, suggesting that they share similar tertiary structures. Surface plasmon resonance (SPR) analysis shows that EDGP binds a soybean 4-kDa hormone-like peptide (4-kDa peptide) in vitro and reduction of EDGP decreased significantly the binding activity, implying that posttranslational modifications are important for its function. Therefore, we investigated the posttranslational modifications in EDGP using mass spectrometry. As the result, six disulfide bonds in EDGP were identified: Cys(70)-Cys(158), Cys(84)-Cys(89), Cys(97)-Cys(113), Cys(100)-Cys(108), Cys(201)-Cys(426), and Cys(332)-Cys(378). In addition, the N-terminal glutamine was cyclized into pyroglutamic acid. All four putative glycosylation sites were occupied by N-linked glycans, which have similar masses of m/z 1171. Finally, measuring the mass of the native protein showed that the posttranslational modifications of EDGP (pI 9.5) involved only disulfide bonds, N-terminal modification, and glycosylation.