Examination of the quality of various force fields and solvation models for the equilibrium simulations of GA88 and GB88

Elucidating the relationship between sequence and conformation is essential for the understanding of functions of proteins. While sharing 88 % sequence identity and differing by only seven residues, GA88 and GB88 have completely different structures and serve as ideal systems for investigating the relationship between sequence and function. Benefiting from the continuous advancement of the computational ability of modern computers, molecular dynamics (MD) simulation is now playing an increasingly important role in the study of proteins. However, the reliability of MD simulations is limited by the accuracy of the force fields and solvent model approximations. In this work, several AMBER force fields (AMBER03, AMBER99SB, AMBER12SB, AMBER14SB, AMBER96) and solvent models (TIP3P, IGB5, IGB7, IGB8) have been employed in the simulations of GA88 and GB88. The statistical results from 19 simulations show that GA88 and GB88 both adopt more compact structures than the native structures. GB88 is more stable than GA88 regardless of the force fields and solvent models utilized. Most of the simulations overestimated the salt bridge interaction. The combination of AMBER14SB force field and IGB8 solvent model shows the best overall performance in the simulations of both GA88 and GB88. AMBER03 and AMBER12SB also yield reasonable results but only in the TIP3P explicit solvent model.     

Cytisine attenuates bone loss of ovariectomy mouse by preventing RANKL‐induced osteoclastogenesis

Postmenopausal Osteoporosis (PMOP) is oestrogen withdrawal characterized of much production and activation by osteoclast in the elderly female. Cytisine is a quinolizidine alkaloid that comes from seeds or other plants of the Leguminosae (Fabaceae) family. Cytisine has been shown several potential pharmacological functions. However, its effects on PMOP remain unknown. This study designed to explore whether Cytisine is able to suppress RANKL‐induced osteoclastogenesis and prevent the bone loss induced by oestrogen deficiency in ovariectomized (OVX) mice. In this study, we investigated the effect of Cytisine on RAW 264.7 cells and bone marrow monocytes (BMMs) derived osteoclast culture system in vitro and observed the effect of Cytisine on ovariectomized (OVX) mice model to imitate postmenopausal osteoporosis in vivo. We found that Cytisine inhibited F‐actin ring formation and tartrate‐resistant acid phosphatase (TRAP) staining in dose‐dependent ways, as well as bone resorption by pit formation assays. For molecular mechanism, Cytisine suppressed RANK‐related trigger RANKL by phosphorylation JNK/ERK/p38‐MAPK, IκBα/p65‐NF‐κB, and PI3K/AKT axis and significantly inhibited these signalling pathways. However, the suppression of PI3K‐AKT‐NFATc1 axis was rescued by AKT activator SC79. Meanwhile, Cytisine inhibited RANKL‐induced RANK‐TRAF6 association and RANKL‐related gene and protein markers such as NFATc1, Cathepsin K, MMP‐9 and TRAP. Our study indicated that Cytisine could suppress bone loss in OVX mouse through inhibited osteoclastogenesis. All data provide the evidence that Cytisine may be a promising agent in the treatment of osteoclast‐related diseases such as osteoporosis.     

Insights on Na+ binding and conformational dynamics in multidrug and toxic compound extrusion transporter NorM

MATE (multidrug and toxic compound extrusion) transporter proteins mediate metabolite transport in plants and multidrug resistance in bacteria and mammals. MATE transporter NorM from Vibrio cholerae is an antiporter that is driven by Na+ gradient to extrude the substrates. To understand the molecular mechanism of Na+‐substrate exchange, molecular dynamics simulation was performed to study conformational changes of both wild‐type and mutant NorM with and without cation bindings. Our results show that NorM is able to bind two Na⁺ ions simultaneously, one to each of the carboxylic groups of E255 and D371 in the binding pocket. Furthermore, this di‐Na⁺ binding state is likely more efficient for conformational changes of NorM_VC toward the inward‐facing conformation than single‐Na⁺ binding state. The observation of two Na⁺ binding sites of NorM_VC is consistent with the previous study that two sites for ion binding (denoted as Na1/Na2 sites) are found in the transporter LeuT and BetP, another two secondary transporters. Taken together, our findings shed light on the structure rearrangements of NorM on Na⁺ binding and enrich our knowledge of the transport mechanism of secondary transporters. Proteins 2014; 82:240–249. © 2013 Wiley Periodicals, Inc.     

NADPH oxidase-derived reactive oxygen species in cardiac pathophysiology

Chronic heart failure, secondary to left ventricular hypertrophy or myocardial infarction, is a condition with increasing morbidity and mortality. Although the mechanisms underlying the development and progression of this condition remain a subject of intense interest, there is now growing evidence that redox-sensitive pathways play an important role. This article focuses on the involvement of reactive oxygen species derived from a family of superoxide-generating enzymes, termed NADPH oxidases (NOXs), in the pathophysiology of ventricular hypertrophy, the accompanying interstitial fibrosis and subsequent heart failure. In particular, the apparent ability of the different NADPH oxidase isoforms to define the response of a cell to a range of physiological and pathophysiological stimuli is reviewed. If confirmed, these data would suggest that independently targeting different members of the NOX family may hold the potential for therapeutic intervention in the treatment of cardiac disease.     

Effluxes of solutes from developing seed coats of Phaseolus vulgaris L. and Vicia faba l.: locating the effect of turgor in a coupled chemiosmotic system

Cells lining the developing seed coats of legumes efflux photosynthates (mostly sucrose) and salts (mostly of potassium) into the apoplast for uptake by the developing embryo. These effluxes increase transiently in response to an increase in turgor in the effluxing cells. Detached coats of developing seed of P. haseolus vulgaris and Vicia faba were used to study the effects of turgor on the rates of efflux, on the membrane potential difference and on the membrane pH difference, using a number of inhibitors and agents which might affect signal cascades involving cytoplasmic calcium concentration. Effluxes were measured by measuring the concentrations of solutes of interest in solution samples placed in halves of detached seed coats, the paired halves serving as control and treated sample where appropriate. It is shown that a number of substances affect sucrose and potassium effluxes differently, and that hypo-osmotic shock depolarizes the efflux cells and acidifies the cytoplasm (in P. vulgaris). It is concluded that sucrose and potassium effluxes, although both are increased by an increase in turgor, are affected by different signal pathways. Further, it is also concluded that the signal that increases the rates of both sucrose efflux (via sucrose-proton antiport) and proton pump acts directly on the antiporter rather than on the pump. There are interesting parallels and contrasts between these processes and those in plants such as the charophyte Lamprothamnium after hypo-osmotic shock.     

Molecular cloning, characterization and expression of a C-type lectin cDNA in Chinese mitten crab, Eriocheir sinensis

C-type lectins are pattern-recognition proteins which are functionally important for pathogen recognition and immune regulation in vertebrates and invertebrates. In this study, a lectin cDNA named as Es-Lectin was cloned and characterized from the Chinese mitten crab, Eriocheir sinensis. The full-length sequence of this Es-Lectin cDNA was 651 bp, including an open reading frame of 483 bp encoding 160 amino acids. The predicted molecular weight of the Es-Lectin was 11.8 kDa. A typical signal peptide of 21 amino acids was deduced at the N-terminus of the predicted protein. This Es-Lectin belongs to a C-type lectin and contains six cysteines, a conserved EPN motif (Glu-Pro-Asn) and an imperfect WND (Trp-Asn-Asp) motif (FND, Phe-Asn-Asp). This Es-Lectin had 55% and 32% identity with other two C-type lectins in E. sinensis, and 29-36% homology with decapods. Although the Es-Lectin was also expressed in gill, hepatopancreas, intestine, muscle and stomach, its expression in haemocytes was the greatest. The expression of Es-Lectins in haemocytes increased at 1.5 h after the Aeromonas hydrophila challenge. After a slight decrease, the Es-Lectin expression in haemocytes significantly increased at 48 h post-challenge. The diverse distribution of Es-Lectin and its enhancement by bacterial challenge indicate that C-type lectins are important in the innate immune response to bacterial infection, and can be activated for innate immune response in crab at the initial stage after pathogen infection.     

Equilibrium unfolding mechanism of chicken muscle triose phosphate isomerase

Triose phosphate isomerase (TIM) was prepared and purified from chicken breast muscle. The equilibrium unfolding of TIM by urea was investigated by following the changes of intrinsic fluorescence and circular dichroism spectroscopy, and the equilibrium thermal unfolding by differential scanning calorimetry (DSC). Results show that the unfolding of TIM in urea is highly cooperative and no folding intermediate was detected in the experimental conditions used. The thermodynamic parameters of TIM during its urea induced unfolding were calculated as DeltaG degrees =3.54 kcal.mol(-1), and m(G) = 0.67 kcal.mol(-1)M(-1), which just reflect the unfolding of dissociated folded monomer to fully unfolded monomer transition, while the dissociation energy of folded dimer to folded monomer is probe silence. DSC results indicate that TIM unfolding follows an irreversible two-state step with a slow aggregation process. The cooperative unfolding ratio, DeltaH(cal)/DeltaH(vH), was measured close to 2, indicating that the two subunits of chicken muscle TIM unfold independently. The van't Hoff enthalpy, DeltaH(vH), was estimated as about 200 kcal.mol(-1). These results support the unfolding mechanism with a folded monomer formation before its tertiary structure and secondary structure unfolding.     

AMPK regulates homeostasis of invasion and viability in trophoblasts by redirecting glucose metabolism: Implications for pre‐eclampsia

Pre‐eclampsia (PE) is deemed an ischemia‐induced metabolic disorder of the placenta due to defective invasion of trophoblasts during placentation; thus, the driving role of metabolism in PE pathogenesis is largely ignored. Since trophoblasts undergo substantial glycolysis, this study aimed to investigate its function and regulatory mechanism by AMPK in PE development. Metabolomics analysis of PE placentas was performed by gas chromatography–mass spectrometry (GC–MS). Trophoblast‐specific AMPKα1‐deficient mouse placentas were generated to assess morphology. A mouse PE model was established by Reduced Uterine Perfusion Pressure, and placental AMPK was modulated by nanoparticle‐delivered A769662. Trophoblast glucose uptake was measured by 2‐NBDG and 2‐deoxy‐d‐[³H] glucose uptake assays. Cellular metabolism was investigated by the Seahorse assay and GC–MS.PE complicated trophoblasts are associated with AMPK hyperactivation due not to energy deficiency. Thereafter, AMPK activation during placentation exacerbated PE manifestations but alleviated cell death in the placenta. AMPK activation in trophoblasts contributed to GLUT3 translocation and subsequent glucose metabolism, which were redirected into gluconeogenesis, resulting in deposition of glycogen and accumulation of phosphoenolpyruvate; the latter enhanced viability but compromised trophoblast invasion. However, ablation of AMPK in the mouse placenta resulted in decreased glycogen deposition and structural malformation. These data reveal a novel homeostasis between invasiveness and viability in trophoblasts, which is mechanistically relevant for switching between the ‘go’ and ‘grow’ cellular programs.

Pre‐eclampsia (PE) is associated with trophoblast AMPK hyperactivation, presumably due to LKB1 phosphorylation, and glucose uptake is consequently increased via trafficking of GLUT3 from the cytosol to the plasma membrane. Such translocation enhances glycolytic flux and redirects glucose metabolic intermediates into gluconeogenesis, resulting in PEP accumulation, which not only benefits cell survival but also suppresses invasion by repressing MMPs, and thus in turn modulates switching between the ‘go’ and ‘grow’ cellular programs.