Combined Effects of Agitation,Macromolecular Crowding,and Interfaces on Amyloidogenesis

Amyloid formation and accumulation is a hallmark of protein misfolding diseases and is associated with diverse pathologies including type II diabetes and Alzheimer''s disease (AD). In vitro, amyloidogenesis is widely studied in conditions that do not simulate the crowded and viscous in vivo environment. A high volume fraction of most biological fluids is occupied by various macromolecules, a phenomenon known as macromolecular crowding. For some amyloid systems (e.g. α-synuclein) and under shaking condition, the excluded volume effect of macromolecular crowding favors aggregation, whereas increased viscosity reduces the kinetics of these reactions. Amyloidogenesis can also be catalyzed by hydrophobic-hydrophilic interfaces, represented by the air-water interface in vitro and diverse heterogeneous interfaces in vivo (e.g. membranes). In this study, we investigated the effects of two different crowding polymers (dextran and Ficoll) and two different experimental conditions (with and without shaking) on the fibrilization of amyloid-β peptide, a major player in AD pathogenesis. Specifically, we demonstrate that, during macromolecular crowding, viscosity dominates over the excluded volume effect only when the system is spatially non homogeneous (i.e. an air-water interface is present). We also show that the surfactant activity of the crowding agents can critically influence the outcome of macromolecular crowding and that the structure of the amyloid species formed may depend on the polymer used. This suggests that, in vivo, the outcome of amyloidogenesis may be affected by both macromolecular crowding and spatial heterogeneity (e.g. membrane turn-over). More generally, our work suggests that any factors causing changes in crowding may be susceptibility factors in AD.     

Electrophoretic Mobility of DNA in Solutions of High Ionic Strength

The free-solution mobilities of small single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) have been measured by capillary electrophoresis in solutions containing 0.01–1.0 M sodium acetate. The mobility of dsDNA is greater than that of ssDNA at all ionic strengths because of the greater charge density of dsDNA. The mobilities of both ssDNA and dsDNA decrease with increasing ionic strength until approaching plateau values at ionic strengths greater than ∼0.6 M. Hence, ssDNA and dsDNA appear to interact in a similar manner with the ions in the background electrolyte. For dsDNA, the mobilities predicted by the Manning electrophoresis equation are reasonably close to the observed mobilities, using no adjustable parameters, if the average distance between phosphate residues (the b parameter) is taken to be 1.7 Å. For ssDNA, the predicted mobilities are close to the observed mobilities at ionic strengths ≤0.01 M if the b-value is taken to be 4.1 Å. The predicted and observed mobilities diverge strongly at higher ionic strengths unless the b-value is reduced significantly. The results suggest that ssDNA strands exist as an ensemble of relatively compact conformations at high ionic strengths, with b-values corresponding to the relatively short phosphate-phosphate distances through space.     

Design of a Phosphorylatable PDZ Domain with Peptide-Specific Affinity Changes

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Highlights? Successful design of phosphorylation sites into a globular protein ? Redesigned PDZ domain phosphorylation is associated with local destabilization ? Phosphorylation switches peptide binding function ? Phosphorylation switches peptide recognition specificity     

Structural Insights into a Wildtype Domain of the Oncoprotein E6 and Its Interaction with a PDZ Domain

The high-risk human papilloma virus (HPV) oncoproteins E6 and E7 interact with key cellular regulators and are etiological agents for tumorigenesis and tumor maintenance in cervical cancer and other malignant conditions. E6 induces degradation of the tumor suppressor p53, activates telomerase and deregulates cell polarity. Analysis of E6 derived from a number of high risk HPV finally yielded the first structure of a wild-type HPV E6 domain (PDB 2M3L) representing the second zinc-binding domain of HPV 51 E6 (termed 51Z2) determined by NMR spectroscopy. The 51Z2 structure provides clues about HPV-type specific structural differences between E6 proteins. The observed temperature sensitivity of the well-folded wild-type E6 domain implies a significant malleability of the oncoprotein in vivo. Hence, the structural differences between individual E6 and their malleability appear, together with HPV type-specific surface exposed side-chains, to provide the structural basis for the different interaction networks reported for individual E6 proteins. Furthermore, the interaction of 51Z2 with a PDZ domain of hDlg was analyzed. Human Dlg constitutes a prototypic representative of the large family of PDZ proteins regulating cell polarity, which are common targets of high-risk HPV E6. Nine C-terminal residues of 51Z2 interact with the second PDZ domain of hDlg2. Surface plasmon resonance in conjunction with the NMR spectroscopy derived complex structure (PDB 2M3M) indicate that E6 residues N-terminal to the canonical PDZ-BM of E6 significantly contribute to this interaction and increase affinity. The structure of the complex reveals how residues outside of the classical PDZ-BM enhance the affinity of E6 towards PDZ domains. Such mechanism facilitates successful competition of E6 with cellular PDZ-binding proteins and may apply to PDZ-binding proteins of other viruses as well.     

Discovery and Characterization of a Small Molecule Inhibitor of the PDZ Domain of Dishevelled

Dishevelled (Dvl) is an essential protein in the Wnt signaling pathways; it uses its PDZ domain to transduce the Wnt signals from the membrane receptor Frizzled to downstream components. Here, we report identifying a drug-like small molecule compound through structure-based ligand screening and NMR spectroscopy and show the compound to interact at low micromolar affinity with the PDZ domain of Dvl. In a Xenopus testing system, the compound could permeate the cell membrane and block the Wnt signaling pathways. In addition, the compound inhibited Wnt signaling and reduced the levels of apoptosis in the hyaloid vessels of eye. Moreover, this compound also suppressed the growth of prostate cancer PC-3 cells. These biological effects suggest that by blocking the PDZ domain of Dvl, the compound identified in our studies effectively inhibits the Wnt signaling and thus provides a useful tool for studies dissecting the Wnt signaling pathways.The Wnt signaling pathways are regulated by a family of secreted Wnt glycoproteins. The canonical Wnt pathway, which is highly conserved, is best understood. In this pathway, Wnt molecules interact with the seven-transmembrane Frizzled (Fz)² proteins (1) by binding to an N-terminal cysteine-rich-domain (2). The signal is then transduced into the cell through an internal sequence of Fz, C-terminal to the seventh transmembrane domain, which binds directly to the PDZ (postsynaptic density-95/discs large/zonula occludens-1) domain of the cytoplasmic protein Dishevelled (Dvl) (3). Dvl then transduces the Wnt signals to downstream components (4). Three Dvl homologs (Dvl-1, -2, and -3) have been identified in humans; all are expressed in both embryonic and adult tissues, including brain, heart, lung, kidney, skeletal muscle, and others (4). Up-regulation and overexpression of Dvl proteins have been reported in many cancers, including those of breast, colon, prostate, mesothelium, and lung (non-small cell) (5–8).The Dvl protein is made up of three conserved domains: an N-terminal DIX domain, a central PDZ domain, and a C-terminal DEP domain (9). The central PDZ domain is of particular interest because of its interaction with Fz and other Wnt pathway proteins (3, 10). The direct interaction between the PDZ domain and Fz peptides is relatively weak, and other factors may play a role to ensure the communication between the two molecules (3). For example, several studies suggest that the DEP domain of Dvl has a membrane-targeting function that may facilitate PDZ-Fz interaction (11–14). However, the weak PDZ-Fz interaction provides an opportunity to block Wnt signaling at the Dvl level by using a small molecule inhibitor. An earlier study in our laboratories used an NMR-assisted virtual ligand screening approach to identify a peptide mimic that can bind to the Dvl PDZ domain (15). We have now used an improved algorithm to conduct an additional structure-based virtual screen of the PDZ domain of Dvl and have discovered a group of drug-like compounds that bind to the PDZ domain with moderate to low micromolar affinity. One of these compounds effectively blocked Wnt signaling in vivo and reduced the growth rate of a prostate cancer cell line.     

Inflammatory Conditions Induce IRES-Dependent Translation of cyp24a1

Rapid alterations in protein expression are commonly regulated by adjusting translation. In addition to cap-dependent translation, which is e.g. induced by pro-proliferative signaling via the mammalian target of rapamycin (mTOR)-kinase, alternative modes of translation, such as internal ribosome entry site (IRES)-dependent translation, are often enhanced under stress conditions, even if cap-dependent translation is attenuated. Common stress stimuli comprise nutrient deprivation, hypoxia, but also inflammatory signals supplied by infiltrating immune cells. Yet, the impact of inflammatory microenvironments on translation in tumor cells still remains largely elusive. In the present study, we aimed at identifying translationally deregulated targets in tumor cells under inflammatory conditions. Using polysome profiling and microarray analysis, we identified cyp24a1 (1,25-dihydroxyvitamin D₃ 24-hydroxylase) to be translationally upregulated in breast tumor cells co-cultured with conditioned medium of activated monocyte-derived macrophages (CM). Using bicistronic reporter assays, we identified and validated an IRES within the 5′ untranslated region (5′UTR) of cyp24a1, which enhances translation of cyp24a1 upon CM treatment. Furthermore, IRES-dependent translation of cyp24a1 by CM was sensitive to phosphatidyl-inositol-3-kinase (PI3K) inhibition, while constitutive activation of Akt sufficed to induce its IRES activity. Our data provide evidence that cyp24a1 expression is translationally regulated via an IRES element, which is responsive to an inflammatory environment. Considering the negative feedback impact of cyp24a1 on the vitamin D responses, the identification of a novel, translational mechanism of cyp24a1 regulation might open new possibilities to overcome the current limitations of vitamin D as tumor therapeutic option.     

Fabrication of Carbon Nanotube High-Frequency Nanoelectronic Biosensor for Sensing in High Ionic Strength Solutions

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) ^1-4 make them good candidates for high sensitivity biosensors. When a charged molecule binds to such a sensor surface, it alters the carrier density⁵ in the sensor, resulting in changes in its DC conductance. However, in an ionic solution a charged surface also attracts counter-ions from the solution, forming an electrical double layer (EDL). This EDL effectively screens off the charge, and in physiologically relevant conditions ~100 millimolar (mM), the characteristic charge screening length (Debye length) is less than a nanometer (nm). Thus, in high ionic strength solutions, charge based (DC) detection is fundamentally impeded^6-8.We overcome charge screening effects by detecting molecular dipoles rather than charges at high frequency, by operating carbon nanotube field effect transistors as high frequency mixers^9-11. At high frequencies, the AC drive force can no longer overcome the solution drag and the ions in solution do not have sufficient time to form the EDL. Further, frequency mixing technique allows us to operate at frequencies high enough to overcome ionic screening, and yet detect the sensing signals at lower frequencies^11-12. Also, the high transconductance of SWNT transistors provides an internal gain for the sensing signal, which obviates the need for external signal amplifier.Here, we describe the protocol to (a) fabricate SWNT transistors, (b) functionalize biomolecules to the nanotube¹³, (c) design and stamp a poly-dimethylsiloxane (PDMS) micro-fluidic chamber¹⁴ onto the device, and (d) carry out high frequency sensing in different ionic strength solutions¹¹.     

Actinin-associated LIM Protein: Identification of a Domain Interaction between PDZ and Spectrin-like Repeat Motifs

PDZ motifs are protein–protein interaction domains that often bind to COOH-terminal peptide sequences. The two PDZ proteins characterized in skeletal muscle, syntrophin and neuronal nitric oxide synthase, occur in the dystrophin complex, suggesting a role for PDZ proteins in muscular dystrophy. Here, we identify actinin-associated LIM protein (ALP), a novel protein in skeletal muscle that contains an NH₂-terminal PDZ domain and a COOH-terminal LIM motif. ALP is expressed at high levels only in differentiated skeletal muscle, while an alternatively spliced form occurs at low levels in the heart. ALP is not a component of the dystrophin complex, but occurs in association with α-actinin-2 at the Z lines of myofibers. Biochemical and yeast two-hybrid analyses demonstrate that the PDZ domain of ALP binds to the spectrin-like motifs of α-actinin-2, defining a new mode for PDZ domain interactions. Fine genetic mapping studies demonstrate that ALP occurs on chromosome 4q35, near the heterochromatic locus that is mutated in fascioscapulohumeral muscular dystrophy.     

Ionic Stress and Osmotic Pressure Induce Different Alterations in the Lipopolysaccharide of a Rhizobium meliloti Strain

A halotolerant strain of Rhizobium meliloti was isolated from nodules of a Melilotus plant growing in a salt marsh in Donana National Park (southwest Spain). This strain, EFB1, is able to grow at NaCl concentrations of up to 500 mM, and no effect on growth is produced by 300 mM NaCl. EFB1 showed alterations on its lipopolysaccharide (LPS) structure that can be related to salt stress: (i) silver-stained electrophoretic profiles showed a different mobility that was dependent on ionic stress but not on osmotic pressure, and (ii) a monoclonal antibody, JIM 40, recognized changes in LPS that were dependent on osmotic stress. Both modifications on LPS may form part of the adaptive mechanism of this bacterium for saline environments.     

Estimation of Aeration and Agitation Conditions in Respect to Oxygen Supply and Ventilation

In inosine fermentation, the yield of the product was closely related to the partial pressure of carbon dioxide in the culture system rather than the bicarbonate ion concentration in the culture liquid. The inhibitory effect of carbon dioxide was restored by the methods of reducing the partial pressure of carbon dioxide lower than a certain level. Both ventilation and chemical absorption were applicable for the restoration of the carbon dioxide inhibition, but ventilation had great advantages over the other method from an industrial view-point. In the estimation of aeration-agitation conditions for the scale-up of submerged fermentation in which carbon dioxide was inhibitory, the rate of aeration was to be established to make sufficient ventilation and overcome this inhibition. A scheme for the procedure for the estimation of aeration-agitation conditions under the consideration of influences of carbon dioxide on submerged fermentation was proposed in this paper.     

An Oligomeric Equilibrium Intermediate as the Precursory Nucleus of Globular and Fibrillar Supramacromolecular Assemblies in a PDZ Domain

The equilibrium unfolding at neutral pH of the third PDZ domain of PSD95, as followed by DSC, is characterized by the presence of an equilibrium intermediate with clear signs of oligomerization. DLS and SEC measurements indicate that at 60-70°C small oligomers populate, showing a typical β-sheet far-UV CD spectrum. These intermediate species lead to the formation of rodlike particulates of ∼12 nm, which remain in solution after 2 weeks incubation and grow until they adopt annular/spherical shapes of ∼50 nm and protofibrils, which are subsequently fully transformed into fibrils. The fibrils can also disaggregate after the addition of 1:1 buffer dilution followed by cooling to room temperature, thus returning to the initial monomeric state. Growth kinetics, as shown by ThT and ANS fluorescence, show that the organization of the different supramacromolecular structures comes from a common nucleation unit, the small oligomers, which organize themselves before reaching the incubation temperature of 60°C. Our experiments point toward the existence of a well-defined reversible, stepwise, and downhill organization of the processes involved in the association-dissociation of the intermediate. We estimate the enthalpy change accompanying the association-dissociation equilibria to be 130 kJ × mol⁻¹. Furthermore, the coalescence under essentially reversible conditions of different kinds of supramacromolecular assemblies renders this protein system highly interesting for biophysical studies aimed at our further understanding of amyloid pathological conditions.     

The Role of Backbone Hydrogen Bonds in the Transition State for Protein Folding of a PDZ Domain

Backbone hydrogen bonds are important for the structure and stability of proteins. However, since conventional site-directed mutagenesis cannot be applied to perturb the backbone, the contribution of these hydrogen bonds in protein folding and stability has been assessed only for a very limited set of small proteins. We have here investigated effects of five amide-to-ester mutations in the backbone of a PDZ domain, a 90-residue globular protein domain, to probe the influence of hydrogen bonds in a β-sheet for folding and stability. The amide-to-ester mutation removes NH-mediated hydrogen bonds and destabilizes hydrogen bonds formed by the carbonyl oxygen. The overall stability of the PDZ domain generally decreased for all amide-to-ester mutants due to an increase in the unfolding rate constant. For this particular region of the PDZ domain, it is therefore clear that native hydrogen bonds are formed after crossing of the rate-limiting barrier for folding. Moreover, three of the five amide-to-ester mutants displayed an increase in the folding rate constant suggesting that the hydrogen bonds are involved in non-native interactions in the transition state for folding.     

Development of a Native Escherichia coli Induction System for Ionic Liquid Tolerance

The ability to solubilize lignocellulose makes certain ionic liquids (ILs) very effective reagents for pretreating biomass prior to its saccharification for biofuel fermentation. However, residual IL in the aqueous sugar solution can inhibit the growth and function of biofuel-producing microorganisms. In E. coli this toxicity can be partially overcome by the heterologous expression of an IL efflux pump encoded by eilA from Enterobacter lignolyticus. In the present work, we used microarray analysis to identify native E. coli IL-inducible promoters and develop control systems for regulating eilA gene expression. Three candidate promoters, PmarR’, PydfO’, and PydfA’, were selected and compared to the IPTG-inducible PlacUV5 system for controlling expression of eilA. The PydfA’ and PmarR’ based systems are as effective as PlacUV5 in their ability to rescue E. coli from typically toxic levels of IL, thereby eliminating the need to use an IPTG-based system for such tolerance engineering. We present a mechanistic model indicating that inducible control systems reduce target gene expression when IL levels are low. Selected-reaction monitoring mass spectrometry analysis revealed that at high IL concentrations EilA protein levels were significantly elevated under the control of PydfA’ and PmarR’ in comparison to the other promoters. Further, in a pooled culture competition designed to determine fitness, the strain containing pPmarR’-eilA outcompeted strains with other promoter constructs, most significantly at IL concentrations above 150 mM. These results indicate that native promoters such as PmarR’ can provide effective systems for regulating the expression of heterologous genes in host engineering and simplify the development of industrially useful strains.     

大鼠MUPP1蛋白PDZ8结构域的生化及晶体结构特性

多重PDZ结构域蛋白1型（MUPP1）是一种存在于上皮细胞和神经细胞内含有13个PDZ结构域的重要支架蛋白.在上皮细胞中,MUPP1蛋白在紧密连接结构的形成和上皮细胞的极化过程中发挥重要作用.而在中枢神经系统中,MUPP1基因的1个提前终止突变导致了其最后12个PDZ结构域的缺失,以及严重的先天性脑积水.此外,MUPP1蛋白的表达水平与酒精依赖性和药物戒断的严重性也具有显著的相关性.因此,对MUPP1蛋白所含的PDZ结构域进行纯化和性质鉴定,将有助于深入研究MUPP1蛋白的功能和分子机制.在本文研究中,利用亲和纯化和分子筛技术,对大鼠来源的MUPP1蛋白的第8个PDZ结构域进行了表达和纯化.多角度激光光散射的数据表明: MUPP1-PDZ8结构域在溶液中为单体,分子量为16.4 kD.圆二色谱结果表明,MUPP1-PDZ8结构域具有较好的二级结构折叠,测得其熔解温度为71.6摄氏度,暗示该PDZ结构域在溶液中非常稳定.最后,MUPP1-PDZ8结构域的晶体结构显示,该结构域属于I 型PDZ 结构域,包含3个α螺旋和6个β折叠.其中GLGL模块、β折叠B上的1 351位亮氨酸,以及α螺旋B上的1 405位异亮氨酸/1 398位组氨酸形成的PDZ结合口袋,可以特异性地与其目标蛋白质的羧基末端相结合.综上所述,本文的研究提供了MUPP1-PDZ8结构域的生化特性,以及该结构域与其目标蛋白质相互作用的分子机制,这将为MUPP1蛋白的功能研究提供生物化学与结构生物学的理论基础.     

Increased Activity of Chromatin-bound Ribonucleic Acid Polymerase from Soybean Hypocotyl with Spermidine and High Ionic Strength

Optimal activity of chromatin-bound RNA polymerase from soybeans is obtained with 1 mm Mn²⁻, but only when high ionic strength or polyamines are included in the medium. Such inclusion does not increase the Mg²⁺ activation of the polymerase, but it does lower the concentration needed for optimum activity from 10 mm to 1 mm. Mg²⁻ activation is inhibited by added Mn²⁺, and the inhibition is relieved by high ionic strength or spermidine. The RNA polymerase with either cation is almost entirely polymerase I at low and high ionic strength as evidenced by insensitivity to α-amanitin. Treatment of soybean seedlings with 2,4-dichlorophenoxyacetic acid does not change these characteristics; although the activity rises 3- to 4-fold.     

Structural Basis of a Key Factor Regulating the Affinity between the Zonula Occludens First PDZ Domain and Claudins

The molecular seal between epithelial cells, called the tight junction (TJ), is built by several membrane proteins, with claudins playing the most prominent role. The scaffold proteins of the zonula occludens family are required for the correct localization of claudins and hence formation of the TJ. The intracellular C terminus of claudins binds to the N-terminal PDZ domain of zonula occludens proteins (PDZ1). Of the 23 identified human claudin proteins, nine possess a tyrosine at the −6 position. Here we show that the claudin affinity for PDZ1 is dependent on the presence or absence of this tyrosine and that the affinity is reduced if the tyrosine is modified by phosphorylation. The PDZ1 β2-β3 loop undergoes a significant conformational change to accommodate this tyrosine. Cell culture experiments support a regulatory role for this tyrosine. Plasticity has been recognized as a critical property of TJs that allow cell remodeling and migration. Our work provides a molecular framework for how TJ plasticity may be regulated.     

通过筛选随机多肽文库研究ZO-1中PDZ1结构域的配体结合特点

选择ZO-1的PDZ1结构域作为研究对象,以酵母双杂交为筛选系统,筛选随机多肽文库和与其它PDZ结构域的配体进行相互作用,阐明ZO-1 PDZ1的配体结合特性.ZO-1 PDZ1识别配体C末端保守的氨基酸序列通式可以表示为：［S/T］［F/Y/W］［V/I/L/C］-COOH、［S/T］［K/R］V-COOH、V［F/Y/W］［L/C］-COOH、EYV-COOH.研究发现ZO-1 PDZ1的配体同时具有3种传统PDZ结构域配体的特点,不同的是其结合配体-1位对芳香族氨基酸具有强烈的偏好性.并且某些PDZ结构域配体的-1位和-3位对结构域与配体相互作用的特异性和亲和力有重要的作用.随后通过生物信息学的方法在Swiss-Prot数据库找到与此识别规律相符合的天然人类蛋白质.根据蛋白质的功能和细胞定位等性质选择10个配体用酵母双杂交验证相互作用.证实的相互作用配体有4个.本研究希望用这样的研究策略建立一种有效的研究蛋白质相互作用的方法,通过在全蛋白质组规模上对含有结合配体保守氨基酸序列的蛋白质的查询,理论上可以找到现有数据库中所有可能与目的结构域结合的潜在配体蛋白,特别是那些筛选cDNA文库不容易获得的低丰度配体.