Important Role In Cell Biology Research Articles

Long-distance dependency combined multi-hop graph neural networks for protein–protein interactions prediction

BackgroundProtein–protein interactions are widespread in biological systems and play an important role in cell biology. Since traditional laboratory-based methods have some drawbacks, such as time-consuming, money-consuming, etc., a large number of methods based on deep learning have emerged. However, these methods do not take into account the long-distance dependency information between each two amino acids in sequence. In addition, most existing models based on graph neural networks only aggregate the first-order neighbors in protein–protein interaction (PPI) network. Although multi-order neighbor information can be aggregated by increasing the number of layers of neural network, it is easy to cause over-fitting. So, it is necessary to design a network that can capture long distance dependency information between amino acids in the sequence and can directly capture multi-order neighbor information in protein–protein interaction network.ResultsIn this study, we propose a multi-hop neural network (LDMGNN) model combining long distance dependency information to predict the multi-label protein–protein interactions. In the LDMGNN model, we design the protein amino acid sequence encoding (PAASE) module with the multi-head self-attention Transformer block to extract the features of amino acid sequences by calculating the interdependence between every two amino acids. And expand the receptive field in space by constructing a two-hop protein–protein interaction (THPPI) network. We combine PPI network and THPPI network with amino acid sequence features respectively, then input them into two identical GIN blocks at the same time to obtain two embeddings. Next, the two embeddings are fused and input to the classifier for predict multi-label protein–protein interactions. Compared with other state-of-the-art methods, LDMGNN shows the best performance on both the SHS27K and SHS148k datasets. Ablation experiments show that the PAASE module and the construction of THPPI network are feasible and effective.ConclusionsIn general terms, our proposed LDMGNN model has achieved satisfactory results in the prediction of multi-label protein–protein interactions.

A general computational framework for the dynamics of single- and multi-phase vesicles and membranes

The dynamics of thin, membrane-like structures are ubiquitous in nature. They play especially important roles in cell biology. Cell membranes separate the inside of a cell from the outside, and vesicles compartmentalize proteins into functional microregions, such as the lysosome. Proteins and/or lipid molecules also aggregate and deform membranes to carry out cellular functions. For example, some viral particles can induce the membrane to invaginate and form an endocytic vesicle that pulls the virus into the cell. While the physics of membranes has been extensively studied since the pioneering work of Helfrich in the 1970's, simulating the dynamics of large scale deformations remains challenging, especially for cases where the membrane composition is spatially heterogeneous. Here, we develop a general computational framework to simulate the overdamped dynamics of membranes and vesicles. We start by considering a membrane with an energy that is a generalized functional of the shape invariants and also includes line discontinuities that arise due to phase boundaries. Using this energy, we derive the internal restoring forces and construct a level set-based algorithm that can stably simulate the large-scale dynamics of these generalized membranes, including scenarios that lead to membrane fission. This method is applied to solve for shapes of single-phase vesicles using a range of reduced volumes, reduced area differences, and preferred curvatures. Our results match well the experimentally measured shapes of corresponding vesicles. The method is then applied to explore the dynamics of multiphase vesicles, predicting equilibrium shapes and conditions that lead to fission near phase boundaries.

G-Protein Coupled Receptors (GPCRs) in Insects-A Potential Target for New Insecticide Development.

G-protein coupled receptors (GPCRs) play important roles in cell biology and insects’ physiological processes, toxicological response and the development of insecticide resistance. New information on genome sequences, proteomic and transcriptome analysis and expression patterns of GPCRs in organs such as the central nervous system in different organisms has shown the importance of these signaling regulatory GPCRs and their impact on vital cell functions. Our growing understanding of the role played by GPCRs at the cellular, genome, transcriptome and tissue levels is now being utilized to develop new targets that will sidestep many of the problems currently hindering human disease control and insect pest management. This article reviews recent work on the expression and function of GPCRs in insects, focusing on the molecular complexes governing the insect physiology and development of insecticide resistance and examining the genome information for GPCRs in two medically important insects, mosquitoes and house flies, and their orthologs in the model insect species Drosophila melanogaster. The tissue specific distribution and expression of the insect GPCRs is discussed, along with fresh insights into practical aspects of insect physiology and toxicology that could be fundamental for efforts to develop new, more effective, strategies for pest control and resistance management.

G-Protein Coupled Receptors (GPCRs): Signaling Pathways, Characterization, and Functions in Insect Physiology and Toxicology.

G-protein-coupled receptors (GPCRs) are known to play central roles in the physiology of many organisms. Members of this seven α-helical transmembrane protein family transduce the extracellular signals and regulate intracellular second messengers through coupling to heterotrimeric G-proteins, adenylate cyclase, cAMPs, and protein kinases. As a result of the critical function of GPCRs in cell physiology and biochemistry, they not only play important roles in cell biology and the medicines used to treat a wide range of human diseases but also in insects’ physiological functions. Recent studies have revealed the expression and function of GPCRs in insecticide resistance, improving our understanding of the molecular complexes governing the development of insecticide resistance. This article focuses on the review of G-protein coupled receptor (GPCR) signaling pathways in insect physiology, including insects’ reproduction, growth and development, stress responses, feeding, behaviors, and other physiological processes. Hormones and polypeptides that are involved in insect GPCR regulatory pathways are reviewed. The review also gives a brief introduction of GPCR pathways in organisms in general. At the end of the review, it provides the recent studies on the function of GPCRs in the development of insecticide resistance, focusing in particular on our current knowledge of the expression and function of GPCRs and their downstream regulation pathways and their roles in insecticide resistance and the regulation of resistance P450 gene expression. The latest insights into the exciting technological advances and new techniques for gene expression and functional characterization of the GPCRs in insects are provided.

Ca2+ -regulated cell migration revealed by optogenetically engineered Ca2+ oscillations.

The ability of a single Ca2+ ion to play an important role in cell biology is highlighted by the need for cells to form Ca2+ signals in the dimensions of space, time, and amplitude. Thus, spatial and temporal changes in intracellular Ca2+ concentration are important for determining cell fate. Optogenetic technology has been developed to provide more precise and targeted stimulation of cells. Here, U2OS cells overexpressing Ca2+ translocating channelrhodopsin (CatCh) were used to mediate Ca2+ influx through blue light illumination with various parameters, such as intensity, frequency, duty cycle, and duration. We identified that several Ca2+‐dependent transcription factors and certain kinases can be activated by specific Ca2+ waves. Using a wound‐healing assay, we found that low‐frequency Ca2+ oscillations increased cell migration through the activation of NF‐κB. This study explores the regulation of cell migration by Ca2+ signals. Thus, we can choose optical parameters to modulate Ca2+ waves and achieve activation of specific signaling pathways. This novel methodology can be applied to clarify related cell‐signaling mechanisms in the future.

Structure and Dynamics of Interfacial Peptides and Proteins from Vibrational Sum-Frequency Generation Spectroscopy.

Proteins at interfaces play important roles in cell biology, immunology, bioengineering, and biomimetic material design. Many biological processes are based on interfacial protein action, ranging from cellular communication to immune responses and the protein-driven mineralization of bone. Despite the importance of interfacial proteins, comparatively little is known about their structure. The standard methods for studying crystalline or solution-phase proteins (X-ray diffraction and NMR spectroscopy) are not well-suited for studying proteins at interfaces, and for these proteins we still lack a corresponding technique that can provide the same level of structural resolution. This is not surprising in view of the challenges involved in probing the structure of proteins within monomolecular films assembled at a very thin interface in situ. Vibrational sum-frequency generation (SFG) spectroscopy has the potential to overcome this challenge and investigate the structure and dynamics of proteins at interfaces at the molecular level with subpicosecond time resolution. While SFG studies were initially limited to simple model peptides, the past decade has seen a dramatic advancement of experimental techniques and data analysis methods that has made it possible to also study interfacial proteins and their folding, binding, orientation, hydration, and dynamics. In this review, we first explain the principles of SFG spectroscopy and the experimental and theoretical methods to measure and analyze protein SFG spectra. Then we give an extensive overview of the interfacial proteins studied to date with SFG. We highlight representative examples to demonstrate recent advances in probing the structure of proteins at the interfaces of liquids, membranes, minerals, and synthetic materials.

RETRACTED ARTICLE: Discrimination of urinary exosomes from microvesicles by lipidomics using thin layer liquid chromatography (TLC) coupled with MALDI-TOF mass spectrometry

Urinary extracellular vesicles (EVs), including microvesicles and exosomes, play several important roles in cell biology and serve as potential biomarkers in various kidney diseases. Although they have differential biophysical properties, specific biomarkers are required to discriminate these EVs during isolation/purification. The present study aimed to define differential lipidome profiles of urinary microvesicles vs. exosomes. Urine samples collected from eight healthy individuals were pooled and underwent lipid extraction using 2:1(v/v) chloroform/methanol. The recovered lipids were resolved by thin layer liquid chromatography (TLC) and analyzed by MALDI-TOF MS. From three and five TLC bands observed in microvesicles and exosomes, respectively, several fatty acids, glycerolipids and phospholipids were identified from both EVs without clear differential patterns. However, their sphingolipid profiles were unique. Ceramide phosphates (CerP), hexosyl sphingoid bases (HexSph), lactosyl ceramides (LacCer), mannosyl di-PI-ceramides (M(IP)2 C), sulfatides hexosyl ceramide (SHexCer) and sulfatides hexoxyl sphingoid bases (SHexSph) were detectable only in urinary exosomes, whereas phosphatidylinositol ceramides (PI-Cer) were detectable only in urinary microvesicles. The presence of CerP only in urinary exosomes was successfully validated by dot blot analysis. Our extensive lipidome analyses of urinary microvesicles vs. exosomes provide potential lipidome markers to discriminate exosomes from microvesicles and may lead to better understanding of EVs biogenesis.

Cell-Compatible Nanoprobes for Imaging Intracellular Phosphatase Activities.

Phosphatases play an important role in cell biology, but only a few probes are suitable for selectively imaging phosphatase activity in live cells, because the current probes require cell fixation or exhibit considerable cytotoxicity. Herein, we show that conjugating a d-peptide to a quinazolinone derivative generates cell-compatible, biostable probes for imaging the phosphatase activity inside live cells. Moreover, our results show that inhibiting ectophosphatases is a critical factor for imaging intracellular phosphatases. As the first example of using selective inhibitors to ensure intracellular function of molecular probes, this work illustrates a facile approach to design molecular probes for profiling the activities of enzymes in a spatial, selective manner in a complicated environment.

Synthetic Receptors for the Recognition and Discrimination of Post-Translationally Methylated Lysines.

Post-translational modifications (PTMs) describe the chemical alteration of proteins after their biosynthesis in ribosomes. PTMs play important roles in cell biology, including the regulation of gene expression, cell-cell interactions and the development of different diseases. A prominent class of PTMs is the side-chain methylation of lysine. For the analysis and discrimination of differently methylated lysines, antibodies are widely used, although methylated peptide and protein targets are known to be particularly difficult to differentiate by antibody-based affinity reagents; an additional challenge can be batch-to-batch reproducibility. The application of mass spectrometry techniques for methyllysine discrimination requires a complex sample preparation procedure and is not suited for working in cells. The desire to overcome the above-mentioned challenges has promoted the development of synthetic receptor molecules that recognise and bind methyllysines. Such "artificial antibodies" are of interest for a number of applications, for example, as reagents in biochemical assays, for the isolation and purification of post-translationally methylated proteins and for the tracking of signalling pathways. Moreover, they offer new approaches in diagnostics and therapy. This review delivers an overview of the broad field of methyllysine binding and covers a wide range of synthetic receptors used for the recognition of methylated lysines, including calixarenes, resorcinarenes, pillararenes, disulfide cyclophanes, cucurbiturils and acyclic receptors.

Biophysical Characterization of Lipid Membranes: Effect of Lipid Head Groups and Tails on Synthetic and Natural Lipid Membranes

Lipid membranes play important roles in cell biology. They serve as a structure that protects cellular organelles and maintains unique cell shapes and also as a regulator that controls cellular functions. Thus, understanding the biophysical characteristics lipid membranes, e.g., fluidity and elasticity, is essential, as membrane proteins in cells often modulate these characteristics to properly control cellular activities. Here, we investigate various lipid head groups and tails by utilizing three different experimental techniques: 1) fluorescence recovery after photobleaching (FRAP), 2) black lipid membrane (BLM) electrophysiology recordings, and 3) atomic force microscopy (AFM). Our results show lipid membranes composed of either negatively charged headgroups or lactocylated headgroups have slower lateral diffusion coefficients and reduced elasticity compared to lipids with neutrally charged headgroups or zwitterionic headgroups. Addition of methyl groups in the acyl chain of the lipids decreases lateral diffusion but does not change elasticity of the membrane. Further, we study the biophysical characteristics of two natural lipid membranes extracted from E. coli and P. furiosus, which have known lipid compositions are distinct from each other, and compare our findings from synthetic lipids to understand the biophysical characteristics of natural membranes. We observed that P. furiosus lipid membranes exhibit a slower lateral diffusion and less elasticity compared with E. coli lipid membranes. From correlation among lateral diffusion, gA lifetime (local bending energy), and elasticity, we found that lipid membranes with slower lateral diffusion shows bigger local bending energy and membrane elastic modulus.

Screening miRNA for Functional Significance by 3D Cell Culture System.

Cell-based assays play important roles in cell biology and drug discovery. 3D cell culture, which allows cells to grow or interact with their surrounding in all three dimensions, provides more physiological information for the in vivo tests. Here, we describe a tunable collagen-based 3D cell culture system based on collagen material crosslinked with transgluminase, to study the function of miR. Methods including gel handling, proliferation assays, gene, and protein expressions in a 3D setting are described.

Pharmacophore-Based 3D-QSAR Modeling, Virtual Screening and Molecular Docking Analysis for the Detection of MERTK Inhibitors with Novel Scaffold.

MERTK plays an important role in cell biology and is correlated with many cancers, such as mantle cell lymphomas, pituitary adenomas, and T-cell acute lympholoblastic leukemia. So identification of new MERTK inhibitors is of extreme importance. In this study, 107 MERTK inhibitors with known activities were gathered to generate a ligand-based pharmacophore model (ADDHH.4), followed by building a 3D-QSAR model, which had high value of coefficient of determination (R(2)=0.9061) and high value of coefficient of determination (Q(2)=0.7442). For the pharmacophore model, two hydrogen bond donors (D), one hydrogen bond receptor (A), and two hydrophobic groups (H) were considered as the key elements contributing to ligand activity. The model then served to search a drug-like database with 1.5 million molecules, and 47832 hits were obtained. Subsequently, docking procedure was applied on these hits, and 840 compounds were obtained through high-throughput virtual screening (HTVS). Standard precision (SP), extra precision (XP) and rule of five were also used in virtual screening protocol. Finally, six candidates were identified as potential MERTK inhibitors, with the docking mode in MERTK analyzed.

Micropillar substrates: a tool for studying cell mechanobiology.

Increasing evidence has shown that mechanical cues from the environment play an important role in cell biology. Mechanotransduction or the study of how cells can sense these mechanical cues, and respond to them, is an active field of research. However, it is still not clear how cells sense and respond to mechanical cues. Thus, new tools are being rapidly developed to quantitatively study cell mechanobiology. Particularly, force measurement tools such as micropillar substrates have provided new insights into the underlying mechanisms of mechanosensing. In this chapter, we provide detailed protocol for fabrication, characterization, functionalization, and use of the micropillar substrates.

Quantitative biology: where modern biology meets physical sciences.

Quantitative methods and approaches have been playing an increasingly important role in cell biology in recent years. They involve making accurate measurements to test a predefined hypothesis in order to compare experimental data with predictions generated by theoretical models, an approach that has benefited physicists for decades. Building quantitative models in experimental biology not only has led to discoveries of counterintuitive phenomena but has also opened up novel research directions. To make the biological sciences more quantitative, we believe a two-pronged approach needs to be taken. First, graduate training needs to be revamped to ensure biology students are adequately trained in physical and mathematical sciences and vice versa. Second, students of both the biological and the physical sciences need to be provided adequate opportunities for hands-on engagement with the methods and approaches necessary to be able to work at the intersection of the biological and physical sciences. We present the annual Physiology Course organized at the Marine Biological Laboratory (Woods Hole, MA) as a case study for a hands-on training program that gives young scientists the opportunity not only to acquire the tools of quantitative biology but also to develop the necessary thought processes that will enable them to bridge the gap between these disciplines.

Integrin-Generated Forces Lead to Streptavidin-Biotin Unbinding in Cellular Adhesions

The interplay between chemical and mechanical signals plays an important role in cell biology, and integrin receptors are the primary molecules involved in sensing and transducing external mechanical cues. We used integrin-specific probes in molecular tension fluorescence microscopy to investigate the pN forces exerted by integrin receptors in living cells. The molecular tension fluorescence microscopy probe consisted of a cyclic Arg-Gly-Asp-D-Phe-Lys(Cys) (cRGDfK(C)) peptide tethered to the terminus of a polyethylene glycol polymer that was attached to a surface through streptavidin-biotin linkage. A fluorescence resonance energy transfer mechanism was used to visualize tension-driven extension of the polymer. Surprisingly, we found that integrin receptors dissociate streptavidin-biotin tethered ligands in focal adhesions within 60 min of cell seeding. Although streptavidin-biotin binding affinity is described as the strongest noncovalent bond in nature, and is ∼106 - 108 times larger than that of integrin-RGD affinity, our results suggest that individual integrin-ligand complexes undergo a marked enhancement in stability when the receptor assembles in the cell membrane. Based on the observation of streptavidin-biotin unbinding, we also conclude that the magnitude of integrin-ligand tension in focal adhesions can reach values that are at least 10 fold larger than was previously estimated using traction force microscopy-based methods.

MiRNA let‐7g regulates skeletal myoblast motility via Pinch‐2

Post-transcriptional regulation of gene expression by RNA-binding proteins and by small non-coding RNAs plays an important role in cell biology. Our previous results show that in murine skeletal myoblasts, the expression of Pinch-2, a focal adhesion remodeling factor that regulates cell motility, is repressed by an RNA-binding protein IMP-2/Igf2bp2. We now show that the expression of Pinch-2 is also regulated by the miRNA let-7g. Let-7g and IMP-2 repress Pinch-2 expression independently of each other. A knock-down of let-7g leads to an increase in Pinch-2 expression, and to a decrease of cell motility, which can be reversed by a simultaneous knock-down of Pinch-2. We conclude that let-7g controls the motility of mouse myoblasts in cell culture by post-transcriptionally regulating the expression of Pinch-2.

Measuring transport properties of cell membranes by a PDMS microfluidic device with controllability over changing rate of extracellular solution

The transport of water and chemicals across cell membranes plays an important role in cell biology and in cryopreservation. In this study, a polydimethylsiloxane (PDMS) microfluidic device with hydrodynamic switching is developed to change the extracellular solution at controllable rates in measuring transport properties of cell membranes. To demonstrate the capability of the PDMS microfluidic device, the volume response of trapped chondrocytes to the change in extracellular osmolality is recorded and analyzed to determine the hydraulic conductivity, one of the transport properties of cell membranes. The results show that hydraulic switching is a convenient and reliable method for controlling the switching time constant of the extracellular solution. The switching time constant increases with the increase in the aspiration rate of the syringe pump, which induces the hydrodynamic switching. The two membrane hydraulic conductivities of fresh chondrocytes, measured at two different switching time constants, are statistically identical (t-test, confidence level=95%), which indicates a high credibility for the method developed in this study. The PDMS device reported here represents the first successful use of hydrodynamic switching in a microfluidic device to control the changing rate of extracellular solutions in the measurement of membrane properties.

Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal.

Lipid peroxidation can be described generally as a process under which oxidants such as free radicals attack lipids containing carbon-carbon double bond(s), especially polyunsaturated fatty acids (PUFAs). Over the last four decades, an extensive body of literature regarding lipid peroxidation has shown its important role in cell biology and human health. Since the early 1970s, the total published research articles on the topic of lipid peroxidation was 98 (1970–1974) and has been increasing at almost 135-fold, by up to 13165 in last 4 years (2010–2013). New discoveries about the involvement in cellular physiology and pathology, as well as the control of lipid peroxidation, continue to emerge every day. Given the enormity of this field, this review focuses on biochemical concepts of lipid peroxidation, production, metabolism, and signaling mechanisms of two main omega-6 fatty acids lipid peroxidation products: malondialdehyde (MDA) and, in particular, 4-hydroxy-2-nonenal (4-HNE), summarizing not only its physiological and protective function as signaling molecule stimulating gene expression and cell survival, but also its cytotoxic role inhibiting gene expression and promoting cell death. Finally, overviews of in vivo mammalian model systems used to study the lipid peroxidation process, and common pathological processes linked to MDA and 4-HNE are shown.

Psi Domain of b3 Integrin Has Endogenous Thiol Isomerase Function and Is a Potential New Target for Anti-Thrombotic Therapy

AbstractAbstract 382 Background:Platelets are critical for maintaining hemostasis, but inappropriate platelet activation can lead to pathogenic thrombosis. Integrin aIIbb3, the most abundant protein on the platelet surface, is a key molecule for platelet aggregation and thrombus formation. The PSI domain of b3 integrin is highly conserved among different species but the function of the PSI domain in the integrin family has not been well defined and the role of this domain in hemostasis and thrombosis is poorly understood. It has been reported that b3 integrin possess PDI activity, which may play a role in integrin activation and platelet aggregation. However, whether the PSI domain of b3 integrin has PDI function is currently unknown. Methods:We generated recombinant protein of PSI domain of mouse b3 integrin. Mouse anti-mouse PSI domain mAbs were generated utilising b3 gene deficient mice (b3−/−) immunized with the recombinant protein. Antibody specificity was determined by flow cytometry and western blot. PDI activity assay of mouse PSI domain and native human b3 integrin was performed using reduced and denatured RNase (rdRNase). The effects of the mAbs on platelet function were measured in vitro using aggregometry and in vivo using intravital microscopy thrombosis model. Results:Analysis of the PSI domain of b3 integrin reveals that it contains two CXXC amino acid sequences (the active site motif of PDI), which are highly conserved in different species. Refolding of rdRNase assay showed that the PSI recombinant protein has endogenous PDI activity. Bacitracin, a well-known PDI inhibitor, inhibited PSI domain PDI function in a dose dependent manner. Four anti-PSI domain monoclonal antibodies (mAbs) were generated and showed different inhibitory effects on PDI function of the recombinant PSI domain and purified human platelet b3 integrin. In vitro and ex-vivo studies showed that anti-PSI antibodies inhibited mouse and human platelet aggregation. Using intravital microscopy we demonstrated that anti-PSI mAbs inhibited mouse platelet aggregation and thrombus formation in laser injury thrombosis model. Conclusions:To the best of our knowledge, this is the first time in which it has been demonstrated that the PSI domain of b3 integrin has endogenous PDI activity, which may play important roles in cell biology of platelets and other cells. Our data suggest that the PSI domain of b3 may be a new target in controlling platelet function and our mAbs may have potential in anti-thrombotic therapy. Disclosures:No relevant conflicts of interest to declare.

Immobilization of carbohydrate clusters on a quartz crystal microbalance sensor surface

The immobilization of carbohydrates on gold surfaces is a prerequisite technology for carbohydrate-related studies, including those of carbohydrate–biomolecule interactions. Glycolipid domains in cell membranes, such as lipid rafts, are thought to play an important role in cell biology through their carbohydrate portions. To understand the recognition of glycolipid domains such as receptors for bacterial toxins and viruses, we immobilized clusters of carbohydrates on a gold surface by using polyamidoamine (PAMAM) dendrimers as a scaffold. The PAMAM dendrimers were adsorbed on the gold-coated surface of a quartz crystal microbalance (QCM) sensor and were observed by means of QCM with dissipation (QCM-D). After adsorption of the PAMAM dendrimers, lysoganglioside-GM1 and 12-aminododecyl-N-acetylglucosaminide (GlcNAc–C12–NH2) were immobilized on the amino groups of PAMAM dendrimers by means of an NH2 cross-linker. Immobilization of the carbohydrates was confirmed by observation of their specific interaction with anti-ganglioside GM1 antibody or wheat germ agglutinin (WGA). Surfaces with different GlcNAc–C12–NH2 cluster sizes and densities were prepared by varying the size of the PAMAM dendrimers or the concentration of GlcNAc–C12–NH2 immobilized on the dendrimers, respectively. Analysis of the binding between the GlcNAc–C12–NH2-immobilized surface and WGA revealed that the size of the PAMAM dendrimers influenced the GlcNAc–C12–NH2–WGA interaction, with larger dendrimers resulting in higher WGA binding constants.