Towards Molecular Understanding of the pH Dependence Characterizing NhaA of Which Structural Fold is Shared by Other Transporters

Na⁺/H⁺ antiporters comprise a super-family (CPA) of membrane proteins that are found in all kingdoms of life and are essential in cellular homeostasis of pH, Na⁺ and volume. Their activity is strictly dependent on pH, a property that underpins their role in pH homeostasis. While several human homologues have long been drug targets, NhaA of Escherichia coli has become the paradigm for this class of secondary active transporters as NhaA crystal structure provided insight into the architecture of this molecular machine. However, the mechanism of the strict pH dependence of NhaA is missing. Here, as a follow up of a recent evolutionary analysis that identified a ‘CPA motif’, we rationally designed three E. coli NhaA mutants: D133S, I134T, and the double mutant D133S-I134T. Exploring growth phenotype, transport activity and Li⁺-binding of the mutants, we revealed that Asp133 does not participate directly in proton binding, nor does it directly dictate the pH-dependent transport of NhaA. Strikingly, the variant I134T lost some of the pH control, and the D133S-Il134T double mutant retained Li⁺ binding in a pH independent fashion. Concurrent to loss of pH control, these mutants bound Li⁺ more strongly than the WT. Both positions are in close vicinity to the ion-binding site of the antiporter, attributing the results to electrostatic interaction between these residues and Asp164 of the ion-binding site. This is consistent with pH sensing resulting from direct coupling between cation binding and deprotonation in Asp164, which applies also to other CPA antiporters that are involved in human diseases.     

Mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid to bovine serum albumin

The mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid (HABA) to bovine serum albumin was studied by relaxation methods as well as the binding isotherm using gel chromatography. A single relaxation was observed over a wide range of HABA concentration except at the extremes of high concentration where another slow process was observed. The concentration dependence of the reciprocal relaxation time of the fast process decreased monotonically with increase in concentration of HABA at constant polymer concentration. The data were analyzed on the basis of Brown's domain structure model and were found to be consistent with a sequential binding mechanism. The azohydrazon tautomerism of HABA was identified with the intramolecular step of the complex. The activation parameters of the step, determined from the temperature dependence of the relaxation time of the fast process, showed that this step is rate limited by an enthalpy barrier in both forward and backward directions. Comparison of the activation parameters with those of other serum albumin-ligand systems suggests that there is an enthalpy-entropy compensation in the activation process of the intramolecular step with the compensation temperature at about 270 K; the enthalpy-entropy compensation is thought to be related to the hydrophobic nature of the ligand.     

Association between FABP2 Ala54Thr polymorphisms and type 2 diabetes mellitus risk: a HuGE Review and Meta‐Analysis

Many studies have examined the association between the FABP2 (rs1799883) Ala54Thr gene polymorphism and type 2 diabetes mellitus risk (T2DM) in various populations, but their results have been inconsistent. To assess this relationship more precisely, A HuGE review and meta‐analysis were performed. The PubMed and CNKI database was searched for case‐control studies published up to April 2014. Data were extracted and pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated. Ultimately, 13 studies, comprising 2020 T2DM cases and 2910 controls were included. Overall, for the Thr carriers (Ala/Thr and Thr/Thr) versus the wild‐type homozygotes (Ala/Ala), the pooled OR was 1.18 (95% CI = 1.04–1.34, P = 0.062 for heterogeneity), for Thr/Thr versus Ala/Ala the pooled OR was 1.17 (95% CI = 1.05–1.41 P = 0.087 for heterogeneity). In the stratified analysis by ethnicity, the significantly risks were found among Asians but not Caucasians. This meta‐analysis suggests that the FABP2 (rs1799883) Ala54Thr polymorphisms are associated with increased susceptibility to T2DM risk among Asians but not Caucasians.     

Structure-wise discrimination of adenine and guanine by proteins on the basis of their nonbonded interactions

We have analyzed the nonbonded interactions of the structurally similar moieties, adenine and guanine forming complexes with proteins. The results comprise (a) the amino acid–ligand atom preferences, (b) solvent accessibility of ligand atoms before and after complex formation with proteins, and (c) preferred amino acid residue atoms involved in the interactions. We have observed that the amino acid preferences involved in the hydrogen bonding interactions vary for adenine and guanine. The structural variation between the purine atoms is clearly reflected by their burial tendency in the solvent environment. Correlation of the mean amino acid preference values show the variation that exists between adenine and guanine preferences of all the amino acid residues. All our observations provide evidence for the discriminating nature of the proteins in recognizing adenine and guanine.     

Multiomic Metabolic Enrichment Network Analysis Reveals Metabolite–Protein Physical Interaction Subnetworks Altered in Cancer

Metabolism is recognized as an important driver of cancer progression and other complex diseases, but global metabolite profiling remains a challenge. Protein expression profiling is often a poor proxy since existing pathway enrichment models provide an incomplete mapping between the proteome and metabolism. To overcome these gaps, we introduce multiomic metabolic enrichment network analysis (MOMENTA), an integrative multiomic data analysis framework for more accurately deducing metabolic pathway changes from proteomics data alone in a gene set analysis context by leveraging protein interaction networks to extend annotated metabolic models. We apply MOMENTA to proteomic data from diverse cancer cell lines and human tumors to demonstrate its utility at revealing variation in metabolic pathway activity across cancer types, which we verify using independent metabolomics measurements. The novel metabolic networks we uncover in breast cancer and other tumors are linked to clinical outcomes, underscoring the pathophysiological relevance of the findings.     

A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer–Associated Stellate Cell Small Extracellular Vesicles

Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor-derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography–tandem mass spectrometry. Most of the 1481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1–like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy.     

Dynamic and Reversible Aggregation of the Human CAP Superfamily Member GAPR-1 in Protein Inclusions in Saccharomyces cerevisiae

Many proteins that can assemble into higher order structures termed amyloids can also concentrate into cytoplasmic inclusions via liquid–liquid phase separation. Here, we study the assembly of human Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1), an amyloidogenic protein of the Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP) protein superfamily, into cytosolic inclusions in Saccharomyces cerevisiae. Overexpression of GAPR-1-GFP results in the formation GAPR-1 oligomers and fluorescent inclusions in yeast cytosol. These cytosolic inclusions are dynamic and reversible organelles that gradually increase during time of overexpression and decrease after promoter shut-off. Inclusion formation is, however, a regulated process that is influenced by factors other than protein expression levels. We identified N-myristoylation of GAPR-1 as an important determinant at early stages of inclusion formation. In addition, mutations in the conserved metal-binding site (His54 and His103) enhanced inclusion formation, suggesting that these residues prevent uncontrolled protein sequestration. In agreement with this, we find that addition of Zn²⁺ metal ions enhances inclusion formation. Furthermore, Zn²⁺ reduces GAPR-1 protein degradation, which indicates stabilization of GAPR-1 in inclusions. We propose that the properties underlying both the amyloidogenic properties and the reversible sequestration of GAPR-1 into inclusions play a role in the biological function of GAPR-1 and other CAP family members.     

A transient species of poly(A)+ RNA detected by a myosin heavy chain cDNA probe in muscle cell culture during terminal differentiation

Primary cell cultures were prepared from breast muscles of 11 day 4 hour-embryonic chicks. Cytoplasmic RNAs were isolated from the cultured cells at various time intervals from day 3 to day 8. A [P32] DNA probe complementary to messenger RNA of myosin heavy chain was used to hybridize with the RNAs after gel electrophoresis. A transient species of polyadenylated RNA with a decreased mobility in electrophoresis was detected during a period of time when contractions of syncytial fibers were first observed.     

Binding of [3H]estradiol- and [3H]H1285-receptor complexes to rabbit uterine chromatin

In the present study we investigated the binding characteristics of estrogen and antiestrogen-receptor complexes to rabbit uterine chromatin. Activated or nonactivated estrogen receptors were partially purified by DEAE-cellulose chromatography using low (1 mM) or high (10 mM) concentrations of sodium molybdate. Activated [³H]estradiol-receptor complexes showed enhanced binding to chromatin acceptor sites unmasked by 1 M, 4 M and 6 M guanidine hydrochloride. We also examined the chromatin-binding characteristics of the estrogen receptors when bound by the high-affinity triphenylethylene antiestrogen, H1285. The acceptor site activity for the [³H]H1285-receptor complexes was markedly decreased at sites unmasked by 4 M and 6 M guanidine hydrochloride. Further, the nonactivated receptor complexes showed very low binding to deproteinized chromatin. The estrogen-receptor chromatin-acceptor sites were tissue specific and saturable. These chromatin acceptor sites differ in their affinity and capacity (number of binding sites per cell) for the estrogen- and antiestrogen-receptor complexes. Thus, we suggest that the differences in the physiological and physicochemical properties of estrogens and antiestrogens may be related to their differential interaction with uterine chromatin subfractions.     

Kinetic and proteolytic identification of heat-induced conformational changes in the urea herbicide binding site of isolated Phaseolus vulgaris chloroplast thylakoids

Kinetic parameters of 3-(3, 4-dichlorophenyl)-1, 1-dimethyl urea (DCMU)-induced inhibition of electron transport in chloroplast thylakoids isolated from Phaseolus vulgaris L. cv. Oregon 1604 were determined from analysis of a convergent, parallel electrical circuit. Through this analogue, the apparent affinity of the purported binding site for DCMU (K₁) and the relative amount of DCMU-insensitive electron transport (v^max₁/v_o) were obtained using a reiterative non-linear least squares curve-fitting procedure. Exposure of thylakoids to heat caused a gradual increase in K₁ (or decrease in the affinity of the thylakoid for DCMU) with an apparent activation energy of 134 kJ mol⁻¹. Tryptic susceptibility of a protein region regulating K₁ also decreased gradually with exposure to 45°C, suggesting that the heat-induced increase in K₁ might be due to a protein conformational change. On the other hand, thylakoid exposure to 45°C resulted in a rapid (<5 min) irreversible increase in v^max_I/v_o, which was also the apparent result of a conformational change in a region of the protein which regulates this function. These results are suggestive of the existence of differential thermal sensitivities of proteins within the thylakoids and, perhaps, of different regions within a single membrane protein.