An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer’s disease) to development of the healthy brain (e.g., synaptic pruning)^1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system⁷. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).     

How Amphipols Embed Membrane Proteins: Global Solvent Accessibility and Interaction with a Flexible Protein Terminus

Amphipathic polymers called amphipols provide a valuable alternative to detergents for keeping integral membrane proteins soluble in aqueous buffers. Here, we characterize spatial contacts of amphipol A8-35 with membrane proteins from two architectural classes: The 8-stranded β-barrel outer membrane protein OmpX and the α-helical protein bacteriorhodopsin. OmpX is well structured in A8-35, with its barrel adopting a fold closely similar to that in dihexanoylphosphocholine micelles. The accessibility of A8-35-trapped OmpX by a water-soluble paramagnetic molecule is highly similar to that in detergent micelles and resembles the accessibility in the natural membrane. For the α-helical protein bacteriorhodopsin, previously shown to keep its fold and function in amphipols, NMR data show that the imidazole protons of a polyhistidine tag at the N-terminus of the protein are exchange protected in the presence of detergent and lipid bilayer nanodiscs, but not in amphipols, indicating the absence of an interaction in the latter case. Overall, A8-35 exhibits protein interaction properties somewhat different from detergents and lipid bilayer nanodiscs, while maintaining the structure of solubilized integral membrane proteins.     

LCP-Tm: An Assay to Measure and Understand Stability of Membrane Proteins in a Membrane Environment

Structural and functional studies of membrane proteins are limited by their poor stability outside the native membrane environment. The development of novel methods to efficiently stabilize membrane proteins immediately after purification is important for biophysical studies, and is likely to be critical for studying the more challenging human targets. Lipidic cubic phase (LCP) provides a suitable stabilizing matrix for studying membrane proteins by spectroscopic and other biophysical techniques, including obtaining highly ordered membrane protein crystals for structural studies. We have developed a robust and accurate assay, LCP-T_m, for measuring the thermal stability of membrane proteins embedded in an LCP matrix. In its two implementations, protein denaturation is followed either by a change in the intrinsic protein fluorescence on ligand release, or by an increase in the fluorescence of a thiol-binding reporter dye that measures exposure of cysteines buried in the native structure. Application of the LCP-T_m assay to an engineered human β₂-adrenergic receptor and bacteriorhodopsin revealed a number of factors that increased protein stability in LCP. This assay has the potential to guide protein engineering efforts and identify stabilizing conditions that may improve the chances of obtaining high-resolution structures of intrinsically unstable membrane proteins.     

Soluble and Insoluble Protein Patterns during Induction of Freezing Tolerance in Black Locust Seedlings

Protein metabolism plays a major role in the development of freezing tolerance in plants. Soluble and insoluble protein concentrations were followed during induction of freezing tolerance in black locust (Robinia pseudoacacia L.) stem tissues. Soluble proteins were fractionated using two-dimensional polyacrylamide gel electrophoresis and examined for the presence of glycoprotein fractions. Soluble protein concentration remained relatively constant during early stages of induction of freezing tolerance but increased significantly during later stages, while insoluble protein concentration remained relatively constant throughout induction. A new soluble protein component appeared during later stages of induction and was identified as a glycoprotein. Some glycoproteins are known to have a high water-binding capacity, which could play a role in intracellular resistance to ice formation during development of freezing tolerance.     

Vaccinia Viral Protein A27 Is Anchored to the Viral Membrane via a Cooperative Interaction with Viral Membrane Protein A17

The vaccinia viral protein A27 in mature viruses specifically interacts with heparan sulfate for cell surface attachment. In addition, A27 associates with the viral membrane protein A17 to anchor to the viral membrane; however, the specific interaction between A27 and A17 remains largely unclear. To uncover the active binding sites and the underlying binding mechanism, we expressed and purified the N-terminal (18–50 residues) and C-terminal (162–203 residues) fragments of A17, which are denoted A17-N and A17-C. Through surface plasmon resonance, the binding affinity of A27/A17-N (K_A = 3.40 × 10⁸ m⁻¹) was determined to be approximately 3 orders of magnitude stronger than that of A27/A17-C (K_A = 3.40 × 10⁵ m⁻¹), indicating that A27 prefers to interact with A17-N rather than A17-C. Despite the disordered nature of A17-N, the A27-A17 interaction is mediated by a specific and cooperative binding mechanism that includes two active binding sites, namely ³²SFMPK³⁶ (denoted as F1 binding) and ²⁰LDKDLFTEEQ²⁹ (F2). Further analysis showed that F1 has stronger binding affinity and is more resistant to acidic conditions than is F2. Furthermore, A27 mutant proteins that retained partial activity to interact with the F1 and F2 sites of the A17 protein were packaged into mature virus particles at a reduced level, demonstrating that the F1/F2 interaction plays a critical role in vivo. Using these results in combination with site-directed mutagenesis data, we established a computer model to explain the specific A27-A17 binding mechanism.     

A Matrix Protein Silences Transposons and Repeats through Interaction with Retinoblastoma-Associated Proteins

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Highlights? The Arabidopsis matrix protein TEK silences transposons and repeat-containing genes ? Binding of TEK on targets affects chromatin conformation and histone modifications ? TEK protein associates with FVE/MSI5-containing histone deacetylation complex ? TEK directs repressive modification as a key structural component in gene silencing     

Covalent Fixation of Soluble Derivatized Dextrans to Model Proteins in Low-Concentration Medium: Application to Factor IX and Protein C

Factor IX and protein C are zymogens implicated in blood clotting, and an increase in their plasmatic residence time would be of interest for the treatment of the disorders caused by their deficiency. In this context, the conjugation of these proteins to polymers such as modified dextrans could be used to approach the problem. Conjugate formation in concentrated medium ([protein]>50 g/L) is well documented, whereas drastic dilution ([protein] <1 g/L) is quite unfavorable. Before studying the binding of factor IX and protein C to polymers, the coupling of model proteins (human hemoglobin, Hb; human serum albumin, HSA) in low-concentration medium to benzenetetracarboxylate dextran (BTC-dextran) and dialdehyde dextran was investigated. To obtain soluble benzenetetracarboxylate dextran-based conjugates, the conditions of coupling were optimized; the use of sulfo-NHS was necessary to form a conjugate with benzenetetracarboxylate dextran. In fact, the O-acylurea intermediate formed between coupling agent [l-ethyl-3(3-dimethylaminopropyl) carbodiimide, EDC] and BTC-dextran must be stabilized. Concerning dialdehyde dextran, a more oxidized polymer and a higher pH of the buffer of coupling than for highly concentrated solution must be used to obtain a conjugate. Whatever polymer is used, HSA appeared clearly less reactive than Hb, which can be attributed to the better reactivity of N-terminal amino groups in this latter protein and to the marked affinity of benzenetetracarboxylate dextran for it. No soluble conjugate was formed between the same dextran derivatives and factor IX or protein C. Moreover, the activity of both coagulation factors was dramatically decreased by contact with EDC and glutaraldehyde, a small molecule. Thus, bad accessibility of protein amino groups is probably responsible for this lack of reactivity. Nevertheless, it could be shown that carboxylate and amino groups were essential to the activity of factor IX and protein C.     

A Rapid and Sensitive Fluorimetric Protocol for the Quantification of Green Fluorescent Protein in Soluble Protein Extracts from Transgenic Arabidopsis

Green fluorescent protein (GFP) is a popular qualitative reporter protein used to study different aspects of plant biology. However, to be used as a reliable quantitative reporter in expression studies using fluorescence based assays, methods to eliminate interfering endogenous molecules must be considered. Therefore, a standard curve based solid phase fluorescent immunoassay that eliminates the effects of interfering endogenous molecules was developed to quantify the GFP levels in soluble green extracts prepared from plants. Microtiter plates coated with anti-GFP were used to capture GFP from soluble plant extracts, interfering endogenous molecules was eliminated by washing without disturbing the anti-GFP binding of GFP, and then the fluorescence intensity of bound GFP was measured using a spectrofluorometer. We report in this study the use of this method to quantify the expression levels of soluble modified GFP in transgenic Arabidopsis thaliana.     

A Soluble Protein Factor is Required in Vitro for Membrane Insertion of the Thylakoid Precursor Protein, pLHCP

The precursor to the light-harvesting chlorophyll a/b protein of photosystem II can insert into isolated thylakoid membranes if reaction mixtures also contain ATP and a soluble extract of chloroplasts. Optimization of this insertion process and the initial characterization of the soluble chloroplastic component are presented. With a fixed amount of precursor, maximum integration rates occurred during the first 30 minutes at pH 8.0 and 30°C when the soluble chloroplast extract was increased eight-fold over the stoichiometric amount. Under these conditions, insertion was routinely about 60% of that which occurred during import into intact chloroplasts. Integration also increased virtually linearly with increasing amounts of precursor. However, assays revealed that at least 40% of the in vitro-synthesized pLHCP was pelletable and inactive. The soluble chloroplastic component exhibited characteristics expected of a protein. It was inactivated by heat, protease, and N-ethylmaleimide, but was insensitive to ribonuclease. The soluble component migrated on a Sephacryl S-200 gel filtration column as a single peak with an M_r of approximately 65,000. The proteinaceous nature of this factor suggests a similarity to soluble factors required for protein transport/integration in other membrane systems.     

Interaction Between Nickel and Protein Source in the Ruminant

Eighty growing steers were used to determine the effect of nickel supplementation on performance and metabolic parameters of steers fed corn silage-based diets supplemented with different crude protein sources. Crude protein sources examined included: (1) soybean meal, (2) blood meal, (3) urea, and (4) blood meal-urea (two-thirds of supplemental nitrogen from blood meal and one-third from urea). The protein sources differed in ruminal degradability, nitrogen solubility, and nickel content. Nickel was added within each protein treatment to supply either 0 or 5 ppm of supplemental nickel. The experiment was 84 d in duration and rumen fluid and blood samples were collected on days 42 and 80. Average daily gain and feed efficiency were not affected by nickel supplementation. The addition of 5 ppm supplemental nickel greatly increased rumen bacterial urease activity regardless of protein source. When samples were collected prior to feeding on day 80, nickel increased serum urea nitrogen concentrations in steers fed urea, but decreased circulating urea concentrations in animals fed blood meal or the blood meal-urea combination.Ad libitum intake of trace mineral salt was greatly reduced in steers receiving 5 ppm supplemental nickel. The present study suggests that the source of protein may influence ruminant responses to dietary nickel.     

A Novel Interaction Between Aging and ER Overload in a Protein Conformational Dementia

Intraneuronal deposition of aggregated proteins in tauopathies, Parkinson disease, or familial encephalopathy with neuroserpin inclusion bodies (FENIB) leads to impaired protein homeostasis (proteostasis). FENIB represents a conformational dementia, caused by intraneuronal polymerization of mutant variants of the serine protease inhibitor neuroserpin. In contrast to the aggregation process, the kinetic relationship between neuronal proteostasis and aggregation are poorly understood. To address aggregate formation dynamics, we studied FENIB in Caenorhabditis elegans and mice. Point mutations causing FENIB also result in aggregation of the neuroserpin homolog SRP-2 most likely within the ER lumen in worms, recapitulating morphological and biochemical features of the human disease. Intriguingly, we identified conserved protein quality control pathways to modulate protein aggregation both in worms and mice. Specifically, downregulation of the unfolded protein response (UPR) pathways in the worm favors mutant SRP-2 accumulation, while mice overexpressing a polymerizing mutant of neuroserpin undergo transient induction of the UPR in young but not in aged mice. Thus, we find that perturbations of proteostasis through impairment of the heat shock response or altered UPR signaling enhance neuroserpin accumulation in vivo. Moreover, accumulation of neuroserpin polymers in mice is associated with an age-related induction of the UPR suggesting a novel interaction between aging and ER overload. These data suggest that targets aimed at increasing UPR capacity in neurons are valuable tools for therapeutic intervention.     

Predicting the Binding Patterns of Hub Proteins: A Study Using Yeast Protein Interaction Networks

Background

Protein-protein interactions are critical to elucidating the role played by individual proteins in important biological pathways. Of particular interest are hub proteins that can interact with large numbers of partners and often play essential roles in cellular control. Depending on the number of binding sites, protein hubs can be classified at a structural level as singlish-interface hubs (SIH) with one or two binding sites, or multiple-interface hubs (MIH) with three or more binding sites. In terms of kinetics, hub proteins can be classified as date hubs (i.e., interact with different partners at different times or locations) or party hubs (i.e., simultaneously interact with multiple partners).

Methodology

Our approach works in 3 phases: Phase I classifies if a protein is likely to bind with another protein. Phase II determines if a protein-binding (PB) protein is a hub. Phase III classifies PB proteins as singlish-interface versus multiple-interface hubs and date versus party hubs. At each stage, we use sequence-based predictors trained using several standard machine learning techniques.

Conclusions

Our method is able to predict whether a protein is a protein-binding protein with an accuracy of 94% and a correlation coefficient of 0.87; identify hubs from non-hubs with 100% accuracy for 30% of the data; distinguish date hubs/party hubs with 69% accuracy and area under ROC curve of 0.68; and SIH/MIH with 89% accuracy and area under ROC curve of 0.84. Because our method is based on sequence information alone, it can be used even in settings where reliable protein-protein interaction data or structures of protein-protein complexes are unavailable to obtain useful insights into the functional and evolutionary characteristics of proteins and their interactions.

Availability

We provide a web server for our three-phase approach: http://hybsvm.gdcb.iastate.edu.     

脆性组氨酸三联体与复制蛋白A相互作用的鉴定

目的：研究脆性组氨酸三联体（Fhit）对ATR／CHKl通路的影响,确定Fhit与复制蛋白A（RPA）存在相互作用,为进一步研究Fhit特异的信号通路奠定基础。方法：将Fhit全长基因插入含GST基因的原核表达载体中,在大肠杆菌中表达纯化GST-Fhit融合蛋白,并用GST沉降技术研究Fhit与RPA是否在体外存在相互作用;在表达Fhit的人细胞中用免疫共沉淀技术分析Fhit与RPA是否在体内存在相互作用,同时用免疫荧光染色方法研究Fhit与RPA在细胞内是否可以共定位。结果：通过免疫共沉淀、免疫荧光染色及GST沉降技术,确定了Fhit与RPA在体内及体外均可以相互作用。结论：确定了Fhit与RPA之间存在相互作用,为阐明Fhit在维持基因组完整性方面的机理提供了线索。     

Fine Mapping of the Interaction between C4b-Binding Protein and Outer Membrane Proteins LigA and LigB of Pathogenic Leptospira interrogans

The complement system consists of more than 40 proteins that participate in the inflammatory response and in pathogen killing. Complement inhibitors are necessary to avoid the excessive consumption and activation of this system on host cells. Leptospirosis is a worldwide zoonosis caused by spirochetes from the genus Leptospira. Pathogenic leptospires are able to escape from complement activation by binding to host complement inhibitors Factor H [FH] and C4b-binding protein (C4BP) while non-pathogenic leptospires are rapidly killed in the presence of fresh serum. In this study, we demonstrate that complement control protein domains (CCP) 7 and 8 of C4BP α-chain interact with the outer membrane proteins LcpA, LigA and LigB from the pathogenic leptospire L. interrogans. The interaction between C4BP and LcpA, LigA and LigB is sensitive to ionic strength and inhibited by heparin. We fine mapped the LigA and LigB domains involved in its binding to C4BP and heparin and found that both interactions are mediated through the bacterial immunoglobulin-like (Big) domains 7 and 8 (LigA7-8 and LigB7-8) of both LigA and LigB and also through LigB9-10. Therefore, C4BP and heparin may share the same binding sites on Lig proteins.     

Membrane Skeletal Proteins and Their Integral Membrane Protein Anchors are Targets for Tyrosine and Threonine Kinases in Euglena

ABSTRACT. Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [³²P]-orthophosphate or γ-[³²P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.     

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography. While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process. Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.). Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment. To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available. To demonstrate this protocol, representative results are presented in which chimeric murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber. Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods.     

Fusion Proteins and Select Lipids Cooperate as Membrane Receptors for the Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE) Vam7p

Vam7p, the vacuolar soluble Q_c-SNARE, is essential for yeast vacuole fusion. The large tethering complex, homotypic fusion and vacuole protein sorting complex (HOPS), and phosphoinositides, which interact with the Vam7p PX domain, have each been proposed to serve as its membrane receptors. Studies with the isolated organelle cannot determine whether these receptor elements suffice and whether ligands or mutations act directly or indirectly on Vam7p binding to the membrane. Using pure components that are active in reconstituted vacuolar fusion, we now find that Vam7p binds to membranes through its combined affinities for several vacuolar membrane constituents: HOPS, phosphatidylinositol 3-phosphate, SNAREs, and acidic phospholipids. Acidic lipids allow low concentrations of Vam7p to suffice for fusion; without acidic lipids, the block to fusion is partially bypassed by high concentrations of Vam7p.     

红颊草莓离体培养及其增殖过程中可溶性蛋白研究

本文以红颊草莓为试材,对红颊草莓茎段进行组织培养,筛选出离体快繁最适的培养基条件,并诱导再生植株,也初步探索了培养物增殖过程中可溶性蛋白的动态变化。结果表明:红颊草莓适宜的诱导分化培养基为MS+6-BA0.5mg/mL+IAA0.25mg/mL,不定芽分化率可达100%;继代增殖最适宜培养基为MS+6-BA0.25mg/L+KT0.25mg/L+IAA0.05mg/L,增殖倍数达到6.44;最佳生根培养基为1/2MS+IAA0.05～0.1mg/L,生根数为5～6条,平均根长2cm左右,且生长健壮;最佳移栽基质为河沙,移栽成活率达88.9%。对红颊草莓增殖过程中的可溶性蛋白研究结果表明:可溶性蛋白含量在第10天、第30天呈现明显的两个高峰,SDS-PAGE电泳结果显示16.7kD、15.6kD和14.4kD的蛋白是草莓生长所必须的,而分子量为26.2kD和23.7kD的蛋白则只在继代增殖旺盛时期出现,为增殖时的特异蛋白组分。本研究结果为红颊草莓快繁育苗提供了一条高效途径,也为进一步研究草莓离体培养过程中体内生理变化的分子机理提供了有意义的参考。