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.     

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.     

Transforming Growth Factor-Beta-Induced Protein (TGFBI)/(βig-H3): A Matrix Protein with Dual Functions in Ovarian Cancer

Transforming growth factor-beta-induced protein (TGFBI, also known as βig-H3 and keratoepithelin) is an extracellular matrix protein that plays a role in a wide range of physiological and pathological conditions including diabetes, corneal dystrophy and tumorigenesis. Many reports indicate that βig-H3 functions as a tumor suppressor. Loss of βig-H3 expression has been described in several cancers including ovarian cancer and promoter hypermethylation has been identified as an important mechanism for the silencing of the TGFBI gene. Our recent findings that βig-H3 is down-regulated in ovarian cancer and that high concentrations of βig-H3 can induce ovarian cancer cell death support a tumor suppressor role. However, there is also convincing data in the literature reporting a tumor-promoting role for βig-H3. We have shown βig-H3 to be abundantly expressed by peritoneal cells and increase the metastatic potential of ovarian cancer cells by promoting cell motility, invasion, and adhesion to peritoneal cells. Our findings suggest that βig-H3 has dual functions and can act both as a tumor suppressor or tumor promoter depending on the tumor microenvironment. This article reviews the current understanding of βig-H3 function in cancer cells with particular focus on ovarian cancer.     

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.     

C-Reactive Protein as a Biomarker for Major Depressive Disorder?

The etiopathogenesis of depression is not entirely understood. Several studies have investigated the role of inflammation in major depressive disorder. The present work aims to review the literature on the association between C-Reactive Protein (CRP) and depression. A systematic review was performed for the topics of ‘CRP’ and ‘depression’ using the PubMed database from inception to December 2021. Fifty-six studies were identified and included in the review. Evidence suggested the presence of dysregulation in the inflammation system in individuals with depression. In most studies, higher blood CRP levels were associated with greater symptom severity, a specific pattern of depressive symptoms, and a worse response to treatment. Moreover, about one-third of depressed patients showed a low-grade inflammatory state, suggesting the presence of a different major depressive disorder (MDD) subgroup with a distinct etiopathogenesis, clinical course, treatment response, and prognosis, which could benefit from monitoring of CRP levels and might potentially respond to anti-inflammatory treatments. This work provides robust evidence about the potential role of CRP and its blood levels in depressive disorders. These findings can be relevant to developing new therapeutic strategies and better understanding if CRP may be considered a valuable biomarker for depression.     

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.     

A Genetically Encoded Diazirine Analogue for RNA–Protein Photo-crosslinking

Ultraviolent crosslinking is a key experimental step in the numerous protocols that have been developed for capturing and dissecting RNA–protein interactions in living cells. UV crosslinking covalently stalls dynamic interactions between RNAs and the directly contacting RNA-binding proteins and enables stringent denaturing downstream purification conditions needed for the enrichment and biochemical analysis of RNA–protein complexes. Despite its popularity, conventional 254 nm UV crosslinking possesses a set of intrinsic drawbacks, with the low photochemical efficiency being the central caveat. Here we show that genetically encoded photoreactive unnatural amino acids bearing a dialkyl diazirine photoreactive group can address this problem. Using the human iron regulatory protein 1 (IRP1) as a model RNA-binding protein, we show that the photoreactive amino acids can be introduced into the protein without diminishing its RNA-binding properties. A sevenfold increase in the crosslinking efficiency compared to conventional 254 nm UV crosslinking was achieved using the diazirine-based unnatural amino acid DiAzKs. This finding opens an avenue for new applications of the unnatural amino acids in studying RNA–protein interactions.     

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.     

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.     

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.     

Is Protein Folding a Thermodynamically Unfavorable,Active, Energy-Dependent Process?

The prevailing current view of protein folding is the thermodynamic hypothesis, under which the native folded conformation of a protein corresponds to the global minimum of Gibbs free energy G. We question this concept and show that the empirical evidence behind the thermodynamic hypothesis of folding is far from strong. Furthermore, physical theory-based approaches to the prediction of protein folds and their folding pathways so far have invariably failed except for some very small proteins, despite decades of intensive theory development and the enormous increase of computer power. The recent spectacular successes in protein structure prediction owe to evolutionary modeling of amino acid sequence substitutions enhanced by deep learning methods, but even these breakthroughs provide no information on the protein folding mechanisms and pathways. We discuss an alternative view of protein folding, under which the native state of most proteins does not occupy the global free energy minimum, but rather, a local minimum on a fluctuating free energy landscape. We further argue that ΔG of folding is likely to be positive for the majority of proteins, which therefore fold into their native conformations only through interactions with the energy-dependent molecular machinery of living cells, in particular, the translation system and chaperones. Accordingly, protein folding should be modeled as it occurs in vivo, that is, as a non-equilibrium, active, energy-dependent process.     

AlphaFold2: A Role for Disordered Protein/Region Prediction?

The development of AlphaFold2 marked a paradigm-shift in the structural biology community. Herein, we assess the ability of AlphaFold2 to predict disordered regions against traditional sequence-based disorder predictors. We find that AlphaFold2 performs well at discriminating disordered regions, but also note that the disorder predictor one constructs from an AlphaFold2 structure determines accuracy. In particular, a naïve, but non-trivial assumption that residues assigned to helices, strands, and H-bond stabilized turns are likely ordered and all other residues are disordered results in a dramatic overestimation in disorder; conversely, the predicted local distance difference test (pLDDT) provides an excellent measure of residue-wise disorder. Furthermore, by employing molecular dynamics (MD) simulations, we note an interesting relationship between the pLDDT and secondary structure, that may explain our observations and suggests a broader application of the pLDDT for characterizing the local dynamics of intrinsically disordered proteins and regions (IDPs/IDRs).     

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。