Prediction of Protein–Protein Interaction with Pairwise Kernel Support Vector Machine

Protein–protein interactions (PPIs) play a key role in many cellular processes. Unfortunately, the experimental methods currently used to identify PPIs are both time-consuming and expensive. These obstacles could be overcome by developing computational approaches to predict PPIs. Here, we report two methods of amino acids feature extraction: (i) distance frequency with PCA reducing the dimension (DFPCA) and (ii) amino acid index distribution (AAID) representing the protein sequences. In order to obtain the most robust and reliable results for PPI prediction, pairwise kernel function and support vector machines (SVM) were employed to avoid the concatenation order of two feature vectors generated with two proteins. The highest prediction accuracies of AAID and DFPCA were 94% and 93.96%, respectively, using the 10 CV test, and the results of pairwise radial basis kernel function are considerably improved over those based on radial basis kernel function. Overall, the PPI prediction tool, termed PPI-PKSVM, which is freely available at http://159.226.118.31/PPI/index.html, promises to become useful in such areas as bio-analysis and drug development.     

Stress-Responsive Expression,Subcellular Localization and Protein–Protein Interactions of the Rice Metacaspase Family

Metacaspases, a class of cysteine-dependent proteases like caspases in animals, are important regulators of programmed cell death (PCD) during development and stress responses in plants. The present study was focused on comprehensive analyses of expression patterns of the rice metacaspase (OsMC) genes in response to abiotic and biotic stresses and stress-related hormones. Results indicate that members of the OsMC family displayed differential expression patterns in response to abiotic (e.g., drought, salt, cold, and heat) and biotic (e.g., infection by Magnaporthe oryzae, Xanthomonas oryzae pv. oryzae and Rhizoctonia solani) stresses and stress-related hormones such as abscisic acid, salicylic acid, jasmonic acid, and 1-amino cyclopropane-1-carboxylic acid (a precursor of ethylene), although the responsiveness to these stresses or hormones varies to some extent. Subcellular localization analyses revealed that OsMC1 was solely localized and OsMC2 was mainly localized in the nucleus. Whereas OsMC3, OsMC4, and OsMC7 were evenly distributed in the cells, OsMC5, OsMC6, and OsMC8 were localized in cytoplasm. OsMC1 interacted with OsLSD1 and OsLSD3 while OsMC3 only interacted with OsLSD1 and that the zinc finger domain in OsMC1 is responsible for the interaction activity. The systematic expression and biochemical analyses of the OsMC family provide valuable information for further functional studies on the biological roles of OsMCs in PCD that is related to abiotic and biotic stress responses.     

Chemical Labeling of Protein 4′-Phosphopantetheinylation

Nature uses a diverse array of protein post-translational modifications (PTMs) to regulate protein structure, activity, localization, and function. Among them, protein 4′-phosphopantetheinylation derived from coenzyme A (CoA) is an essential PTM for the biosynthesis of fatty acids, polyketides, and nonribosomal peptides in prokaryotes and eukaryotes. To explore its functions, various chemical probes mimicking the natural structure of 4′-phosphopantetheinylation have been developed. In this minireview, we summarize these chemical probes and describe their applications in direct and metabolic labeling of proteins in bacterial and mammalian cells.     

Protein Kinases and Parkinson’s Disease

Currently, the lack of new drug candidates for the treatment of major neurological disorders such as Parkinson’s disease has intensified the search for drugs that can be repurposed or repositioned for such treatment. Typically, the search focuses on drugs that have been approved and are used clinically for other indications. Kinase inhibitors represent a family of popular molecules for the treatment and prevention of various cancers, and have emerged as strong candidates for such repurposing because numerous serine/threonine and tyrosine kinases have been implicated in the pathobiology of Parkinson’s disease. This review focuses on various kinase-dependent pathways associated with the expression of Parkinson’s disease pathology, and evaluates how inhibitors of these pathways might play a major role as effective therapeutic molecules.     

Analysis of Protein–Protein Interactions in MCF-7 and MDA-MB-231 Cell Lines Using Phthalic Acid Chemical Probes

Phthalates are a class of plasticizers that have been characterized as endocrine disrupters, and are associated with genital diseases, cardiotoxicity, hepatotoxicity, and nephrotoxicity in the GeneOntology gene/protein database. In this study, we synthesized phthalic acid chemical probes and demonstrated differing protein–protein interactions between MCF-7 cells and MDA-MB-231 breast cancer cell lines. Phthalic acid chemical probes were synthesized using silicon dioxide particle carriers, which were modified using the silanized linker 3-aminopropyl triethoxyslane (APTES). Incubation with cell lysates from breast cancer cell lines revealed interactions between phthalic acid and cellular proteins in MCF-7 and MDA-MB-231 cells. Subsequent proteomics analyses indicated 22 phthalic acid-binding proteins in both cell types, including heat shock cognate 71-kDa protein, ATP synthase subunit beta, and heat shock protein HSP 90-beta. In addition, 21 MCF-7-specific and 32 MDA-MB-231 specific phthalic acid-binding proteins were identified, including related proteasome proteins, heat shock 70-kDa protein, and NADPH dehydrogenase and ribosomal correlated proteins, ras-related proteins, and members of the heat shock protein family, respectively.     

A “Quenchergenic” Chemoselective Protein Labeling Strategy

Dynamic changes in protein structure can be monitored by using a fluorescent probe and a dark quencher. This approach is contingent upon the ability to precisely introduce a fluorophore/quencher pair into two specific sites of a protein of interest. Despite recent advances, there is continued demand for new and convenient approaches to site-selectively label proteins with such optical probes. We have recently developed a chemoselectively rapid azo-coupling reaction (CRACR) for site-specific protein labeling; it relies on rapid coupling between a genetically encoded 5-hydroxytryptophan residue and various aromatic diazonium ions. Herein, it is reported that the product of this conjugation reaction, a highly chromophoric biarylazo group, is a potent fluorescence quencher. The absorption properties of this azo product can be tuned by systematically altering the structure of the aryldiazonium species. A particular “quenchergenic” aryldiazonium has been identified that, upon conjugation, efficiently quenches the fluorescence of green fluorescent protein, which is a widely used genetically encoded fluorescent probe that can be terminally attached to target proteins. This fluorophore/quencher pair was used to evaluate the protein-labeling kinetics of CRACR, as well as to monitor the proteolysis of a fusion protein.     

葡聚糖接枝型Protein A介质的层析性能提升

Sepharose 4FF微球经环氧活化后与葡聚糖溶液反应,得葡聚糖接枝型琼脂糖微球,再经环氧活化和偶联耐碱型Protein A配基,得葡聚糖接枝型高载量Protein A介质,测定了介质在线清洗稳定性能,并进行了热力学研究.结果表明,与常规Protein A介质相比,葡聚糖接枝型Protein A介质的最高流速提高约32%,对抗体h Ig G的动态载量为60.6 mg/m L,分别为常规介质和Mab Select Su Re介质载量的123%和95%;经40次清洗后,葡聚糖接枝型Protein A介质动态载量为原始载量的92%,远高于常规介质的84%,与Mab Select Su Re稳定性基本一致.3种介质对抗体的结合均为熵驱动过程,葡聚糖接枝型Protein A介质的吸附热介于Mab Select Su Re和常规Protein A介质之间.     

Protein engineering of α/β-hydrolase fold enzymes

The superfamily of α/β-hydrolase fold enzymes is one of the largest known protein families, including a broad range of synthetically useful enzymes such as lipases, esterases, amidases, hydroxynitrile lyases, epoxide hydrolases and dehalogenases. This minireview covers methods developed for efficient protein engineering of these enzymes. Special emphasis is placed on the alteration of enzyme properties such as substrate range, thermostability and enantioselectivity for their application in biocatalysis. In addition, concepts for the investigation of the evolutionary relationship between the different members of this protein superfamily are covered, together with successful examples.     

Protein forming — a novel shaping technique for ceramics

A new consolidation method for the forming of ceramics using globular proteins as consolidators/binders was developed. Various protein products: bovine serum albumin (BSA), albumen (egg white powder) and whey protein concentrate (WPC), were shown to work as gelling agents, which was confirmed by rheological measurements and forming experiments. Suspensions of Si₃N₄, Al₂O₃ and ZrO₂ powders with various solids loadings (44–55 vol.%) were prepared, proteins were added and the resulting slips were poured into simply shaped moulds and consolidation (gelling) took place through heating at 80°C. Through an optimisation of the process, it was possible to sinter to high density values (>99% of theoretical) using gas pressure sintering (GPS) of Si₃N₄ materials, and using pressureless sintering in air of the oxides. Among the globular proteins studied, WPC showed the most favourable properties with less serious slip thickening, limited foam formation, faster gelling, sufficiently high green body strength to make demoulding in wet state possible and less cracking during drying. However, a larger amount of WPC was needed to achieve a proper gelling compared with the other proteins. This negatively influenced the sintering conditions by lower density of shaped bodies, which required ceramic powders that sinter more easily to full density. The most critical steps in the processing were to efficiently remove air, to break foam formations, and to avoid cracking during the demoulding and drying of the cast bodies. None of the proteins gave rise to critical stresses during the burnout operation and sintering was carried out without deformation or cracking.     

Peroxisome Proliferator-Activated Receptor α Agonists Regulate Cholesterol Ester Transfer Protein

Peroxisome proliferator-activated receptor alpha (PPARalpha) belongs to the nuclear receptor superfamily that regulates multiple target genes involved in lipid metabolism. Cholesterol ester transfer protein (CETP) is a secreted glycoprotein that modifies high-density lipoprotein (HDL) particles. In humans, plasma CETP activity is inversely correlated with HDL cholesterol levels. We report here that PPARalpha agonists increase CETP mRNA, protein and accordingly its activity. In a human CETP transgenic animal model harboring the natural flanking regions (Jiang et al. in J Clin Investigat 90:1290-1295, 1992), both fenofibrate and a specific synthetic PPARalpha agonist LY970 elevated human CETP mRNA in liver, serum protein and CETP activity. In hamsters, the endogenous liver CETP mRNA level and the serum CETP activity were dose-dependently upregulated by fenofibrate. In addition Wy14643, a PPARalpha agonist, also significantly elevated CETP mRNA and activity. In a carcinoma cell line of hepatic origin, HepG2 cells, overexpression of PPARalpha resulted in increased CETP mRNA and agonist treatment further elevated CETP mRNA levels. We conclude that PPARalpha agonists upregulate CETP expression and activity and may play an important role in PPARalpha (agonist mediated HDL cholesterol homeostasis in humans.     

葡聚糖接枝型耐碱Protein A介质的层析性能提升研究

Sepharose 4FF微球经环氧活化后与葡聚糖溶液反应,得葡聚糖接枝型琼脂糖微球,再经环氧活化和偶联耐碱型Protein A配基,得葡聚糖接枝型高载量Protein A介质,测定了介质在线清洗稳定性能,并进行了热力学研究. 结果表明,与常规Protein A介质相比,葡聚糖接枝型Protein A介质的最高流速提高约32%,对抗体hIgG的动态载量为60.6 mg/mL,分别为常规介质和MabSelect SuRe介质载量的123%和95%;经40次清洗后,葡聚糖接枝型Protein A介质动态载量为原始载量的92%,远高于常规介质的84%,与MabSelect SuRe稳定性基本一致. 3种介质对抗体的结合均为熵驱动过程,葡聚糖接枝型Protein A介质的吸附热介于MabSelect SuRe和常规Protein A介质之间.     

Preparation and Surface Activities of Modified Soy Protein–Dextrin Surfactants

A series of polymeric surfactants has been prepared through the reaction of soy protein with polyethoxylated stearyl ethers of various hydrophilic chain lengths. These surfactants exhibited surface activity, evaluated using surface tension, foaming, and wetting power that was superior to that of traditional surfactants containing only one hydrophobic moiety and one hydrophilic head group. Changing the ethoxylate (EO) group length had a significant effect on the surface activity. Increasing the EO group length decreased the critical micelle concentration (CMC) and increased the surface tension at the CMC (γ_CMC). The good surface properties of these polysaccharide/protein‐type surfactants suggest that they could be used as emulsifiers to prepare oil‐in‐water emulsions displaying good stability.     

一种可用于大规模单克隆抗体纯化的新型Protein A填料

目的比较不同型号Protein A填料的性能,筛选合适的Protein A填料,用于大规模单克隆抗体(简称单抗)纯化。方法采用表达H02单抗的CHO细胞培养上清,测试不同供应商Protein A填料的蛋白结合载量,选择合适的填料,用于大规模单抗纯化;检测宿主细胞蛋白(host cell protein,HCP)、外源DNA及Protein A残留量,以评价其纯化效果;检测Protein A柱在位清洗(cleaning in place,CIP)效果,并验证其循环使用次数。结果 Amsphere Protein A JWT203填料为合适的ProteinA填料,H02单抗大规模纯化后,可有效去除HCP、DNA及脱落Protein A;采用0.1 mol/L NaOH进行CIP,经过300个循环,对H02单抗仍保持80%以上动态结合载量,Protein A脱落无明显增加,对HCP、外源DNA、聚合体的去除能力无明显改变,层析图谱与初始基本一致,Amsphere Protein A JWT203填料的理化性质较稳定。结论 AmsphereProtein A JWT203是一种可用于大规模单抗纯化的新型Protein A填料。     

Protein Spin Labeling with a Photocaged Nitroxide Using Diels–Alder Chemistry

EPR spectroscopy of diamagnetic bio-macromolecules is based on site-directed spin labeling (SDSL). Herein, a novel labeling strategy for proteins is presented. A nitroxide-based spin label has been developed and synthesized that can be ligated to proteins by an inverse-electron-demand Diels–Alder (DA_inv) cycloaddition to genetically encoded noncanonical amino acids. The nitroxide moiety is shielded by a photoremovable protecting group with an attached tetra(ethylene glycol) unit to achieve water solubility. SDSL is demonstrated on two model proteins with the photoactivatable nitroxide for DA_inv reaction (PaNDA) label. The strategy features high reaction rates, combined with high selectivity, and the possibility to deprotect the nitroxide in Escherichia coli lysate.     

Prosopis farcta Seeds: Potential Source of Protein and Unsaturated Fatty Acids?

The goal of this study is to evaluate the chemical composition of the Prosopis farcta seeds. The Kjeldahl method revealed that the level of the total protein was ca. 18 % on a dry weight basis (DW). Gas chromatographic analysis revealed the presence of six fatty acids. Linoleic acid with 57.55 % was the major fatty acid, followed by oleic and palmitic acids (24.58 and 12.91 %, respectively). Total phenolic content was 1.71 mg GAE/g DW (GAE = gallic acid equivalents). Methanolic extracts showed important antioxidant properties. This work highlights the importance of P. farcta seeds as natural and inexpensive sources of protein, unsaturated fatty acid, and phenolic compounds for both cosmetic and pharmaceutical uses.     

GroEL-Assisted Protein Folding: Does It Occur Within the Chaperonin Inner Cavity?

The folding of protein molecules in the GroEL inner cavity under the co-chaperonin GroES lid is widely accepted as a crucial event of GroEL-assisted protein folding. This review is focused on the data showing that GroEL-assisted protein folding may proceed out of the complex with the chaperonin. The models of GroEL-assisted protein folding assuming ligand-controlled dissociation of nonnative proteins from the GroEL surface and their folding in the bulk solution are also discussed.     

3种不同Protein A亲和层析填料纯化抗CD52单克隆抗体的比较

目的比较3种Protein A亲和层析填料MabSelect SuRe、Protein A Diamond及UniMab 50纯化抗CD52单克隆抗体的载量及纯化效果。方法将浓度为1.24 mg/mL的抗CD52单克隆抗体分别流经3种Protein A亲和层析填料,确保保留时间一致,检测流穿液的抗体浓度,以流穿液中抗体浓度达10%上样浓度时的上样量确定填料的最大动态载量。3种填料均按80%最大载量上样,纯化3批抗CD52单克隆抗体培养上清样品,比较洗脱体积、抗体回收率及洗脱收集液中抗体浓度、抗体纯度、电荷异质性、宿主蛋白残余量、宿主DNA残余量、填料配基Protein A脱落量。结果填料MabSelect SuRe与Protein A Diamond在载量、宿主蛋白残余量、填料配基Protein A脱落量方面接近;在抗体纯度、电荷异质性、宿主DNA残余量方面,3种填料表现相当;在抗体回收率、洗脱体积及抗体浓度方面,UniMab 50表现最佳。结论 3种填料亲和纯化抗CD52单克隆抗体的效果各有优劣,但均可满足该纯化步骤的要求,可根据实际工艺状况及质控要求进行选择。     

Developing an Antibody–Drug Conjugate Approach to Selective Inhibition of an Extracellular Protein

Antibody–drug conjugates (ADCs) are a growing class of therapeutics that harness the specificity of antibodies and the cell-killing potency of small-molecule drugs. Beyond cytotoxics, there are few examples of the application of an ADC approach to difficult drug discovery targets. Here, we present the initial development of a non-internalising ADC, with a view to selectively inhibiting an extracellular protein. Employing the wellinvestigated matrix metalloproteinase-9 (MMP-9) as our model, we adapted a broad-spectrum, nonselective MMP inhibitor for conjugation and linked this to a MMP-9-targeting antibody. The resulting ADC fully inhibits MMP-9, and ELISA results suggest antibody targeting can direct a nonselective inhibitor.     

Diacylglycerol Kinase η Activity in Cells Using Protein Myristoylation and Cellular Phosphatidic Acid Sensor

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to produce phosphatidic acid (PtdOH) and regulates the balance between two lipid second messengers: diacylglycerol and PtdOH. Several lines of evidence suggest that the η isozyme of DGK is involved in the pathogenesis of bipolar disorder. However, the detailed molecular mechanisms regulating the pathophysiological functions remain unclear. One reason is that it is difficult to detect the cellular activity of DGKη. To overcome this difficulty, we utilized protein myristoylation and a cellular PtdOH sensor, the N-terminal region of α-synuclein (α-Syn-N). Although DGKη expressed in COS-7 cells was broadly distributed in the cytoplasm, myristoylated (Myr)-AcGFP-DGKη and Myr-AcGFP-DGKη-KD (inactive (kinase-dead) mutant) were substantially localized in the plasma membrane. Moreover, DsRed monomer-α-Syn-N significantly colocalized with Myr-AcGFP-DGKη but not Myr-AcGFP-DGKη-KD at the plasma membrane. When COS-7 cells were osmotically shocked, all DGKη constructs were exclusively translocated to osmotic shock-responsive granules (OSRG). DsRed monomer-α-Syn-N markedly colocalized with only Myr-AcGFP-DGKη at OSRG and exhibited a higher signal/background ratio (3.4) than Myr-AcGFP-DGKη at the plasma membrane in unstimulated COS-7 cells (2.5), indicating that α-Syn-N more effectively detects Myr-AcGFP-DGKη activity in OSRG. Therefore, these results demonstrated that the combination of myristoylation and the PtdOH sensor effectively detects DGKη activity in cells and that this method is convenient to examine the molecular functions of DGKη. Moreover, this method will be useful for the development of drugs targeting DGKη. Furthermore, the combination of myristoylation (intensive accumulation in membranes) and α-Syn-N can be applicable to assays for various cytosolic PtdOH-generating enzymes.