Identification of chikungunya virus interacting proteins in mammalian cells

Identification and characterization of virus host interactions is an essential step for the development of novel antiviral strategies. Very few studies have been targeted towards identification of chikungunya virus (CHIKV) interacting host proteins. In current study, virus overlay protein binding assay (VOPBA) and matrix-assisted laser desorption/ionization time of flight analysis (MALDI TOF/TOF) were employed for the identification of CHIKV binding proteins in mammalian cells. HSP70 and actin were identified as virus binding proteins in HEK-293T and Vero-E6 cells, whereas STAT-2 was identified as an additional protein in Vero-E6 cells. Pre-incubation with anti-HSP70 antibody and miRNA silencing of HSP70 significantly reduced the CHIKV production in HEK-293T and Vero-E6 cells at early time points. These results suggest that CHIKV exploits the housekeeping molecules such as actin, HSP70 and STAT-2 to establish infection in the mammalian cells.     

The mammalian centromere organization

Routine and recently obtained data on the pattern and functions of the mammalian centromeres and kinetochores have been reviewed. Several problems of kinetochore formation (centromere recognition, anaphase checkpoint) are specially discussed, in addition to the role played by centromere DNA in the interphase nucleus consideration.     

Capture of proteins from mammalian cells in pilot scale using different STREAMLINE adsorbents

This presentation compares three different expanded bed matrices. STREAMLINE rProtein A, STREAMLINE SP-XL and STREAMLINE Chelating were monitored in respect to their ability to clarify the broth, to concentrate and to purify the distinct target protein. The capture of a mouse IgG₁ and a recombinant prothrombin (PT) was carried out in pilot scale using a 100-l bioreactor and STREAMLINE 100 and 200 columns, respectively. The robustness of the process was also estimated monitoring the expansion behaviour and the cell and debris concentrations during the load and in the eluat. In all cases the capture of the target proteins was comparable to conventional chromatographic systems. The purification success was mainly dependent on the selectivity of the ligand used. The affinity process resulted in a highly purified product. The ion exchanger and chelating material mainly concentrated the product. In all three cases 100 l of cell broth were successfully processed in one run. The robustness of the ion exchanger process was poor, because of strong cell matrix interaction. However, for the chelating and especially for the affinity matrix a highly reproducible process was obtained.     

The structure of the mammalian centromere

The mammalian centromere is a multifunctional chromosomal domain with a complexity that is reflected in its higher order structure, DNA sequence organization and protein composition. The centromere plays a major role during cell division where it functions as the site for the integration of the chromosome with the mitotic spindle, the site of the mechanochemical motor responsible for the movement of chromosomes and the major and last point of interaction between sister chromatids. Recent studies have focused on characterizing the components of the centromere and establishing their relationship to its function. The following brief review summarizes some selected aspects of this recent work.     

Identification of prolyl hydroxylation modifications in mammalian cell proteins

Prolyl hydroxylation is a PTM that plays an important role in the formation of collagen fibrils and in the oxygen‐dependent regulation of hypoxia inducible factor‐α (HIF‐α). While this modification has been well characterized in the context of these proteins, it remains unclear to what extent it occurs in the remaining mammalian proteome. We explored this question using MS to analyze cellular extracts subjected to various fractionation strategies. In one strategy, we employed the von Hippel Lindau tumor suppressor protein, which recognizes prolyl hydroxylated HIF‐α, as a scaffold for generating hydroxyproline capture reagents. We report novel sites of prolyl hydroxylation within five proteins: FK506‐binding protein 10, myosin heavy chain 10, hexokinase 2, pyruvate kinase, and C‐1 Tetrahydrofolate synthase. Furthermore, we show that identification of prolyl hydroxylation presents a significant technical challenge owing to widespread isobaric methionine oxidation, and that manual inspection of spectra of modified peptides in this context is critical for validation.     

Identification of novel sphingolipid-binding motifs in mammalian membrane proteins

Specific interactions between transmembrane proteins and sphingolipids is a poorly understood phenomenon, and only a couple of instances have been identified. The best characterized example is the sphingolipid-binding motif VXXTLXXIY found in the transmembrane helix of the vesicular transport protein p24. Here, we have used a simple motif-probability algorithm (MOPRO) to identify proteins that contain putative sphingolipid-binding motifs in a dataset comprising proteomes from mammalian organisms. From these motif-containing candidate proteins, four with different numbers of transmembrane helices were selected for experimental study: i) major histocompatibility complex II Q alpha chain subtype (DQA1), ii) GPI-attachment protein 1 (GAA1), iii) tetraspanin-7 TSN7, and iv), metabotropic glutamate receptor 2 (GRM2). These candidates were subjected to photo-affinity labeling using radiolabeled sphingolipids, confirming all four candidate proteins as sphingolipid-binding proteins. The sphingolipid-binding motifs are enriched in the 7TM family of G-protein coupled receptors, predominantly in transmembrane helix 6. The ability of the motif-containing candidate proteins to bind sphingolipids with high specificity opens new perspectives on their respective regulation and function.     

Identification of proteins released by mammalian cells that mediate DNA internalization through proteoglycan-dependent macropinocytosis

Naked DNA plasmid represents the simplest vehicle for gene therapy and DNA-based vaccination purposes; however, the molecular mechanisms of DNA uptake in mammalian cells are poorly understood. Here, we show that naked DNA uptake occurs via proteoglycan-dependent macropinocytosis, thus challenging the concept of a specific DNA-internalizing receptor. Cells genetically deficient in proteoglycans, which constitute a major source of cell-surface polyanions, exhibited substantially decreased uptake of likewise polyanionic DNA. The apparent paradox was explained by the action of DNA-transporting proteins present in conditioned medium. Complexes between these proteins and DNA require proteoglycans for cellular entry. Mass spectrometry analysis of cell medium components identified several proteins previously shown to associate with DNA and to participate in membrane transport of macromolecular cargo. The major pathway for proteoglycan-dependent DNA uptake was macropinocytosis, whereas caveolae-dependent and clathrin-dependent pathways were not involved, as determined by using caveolin-1 knock-out cells, dominant-negative constructs for dynamin and Eps15, and macropinocytosis-disruptive drugs, as well as confocal fluorescence co-localization studies. Importantly, a significant fraction of internalized DNA was translocated to the nucleus for expression. Our results provide novel insights into the mechanism of DNA uptake by mammalian cells and extend the emerging role of proteoglycans in macromolecular transport.     

Identification and characterization of calmodulin-binding proteins in mammalian sperm flagella

A calcium and calmodulin-regulated cyclic nucleotide phosphodiesterase has been shown to be an integral component of both rat and bovine sperm flagella. The calcium-activated enzyme was inhibited by both trifluoperazine (ID50 = 10 microM) and [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid (EGTA), and the basal activity measured in the presence of EGTA was stimulated by limited proteolysis to that observed in the presence of calcium/calmodulin. 125I-Calmodulin binding to purified rat sperm flagella has been characterized and the flagellar-associated calmodulin-binding proteins identified by a combination of gel and nitrocellulose overlay procedures and by chemical cross-linking experiments using dimethyl suberimidate. 125I-Calmodulin bound to demembranated rat sperm flagella in a time- and concentration-dependent manner. At equilibrium, 30-40% of the bound 125I-calmodulin remains associated with the flagella after treatment with EGTA or trifluoperazine. The majority of the bound 125I-calmodulin, both the Ca2+-dependent and -independent, was displaced by excess calmodulin. A 67-kDa calmodulin-binding protein was identified by both the gel and nitrocellulose overlay procedures. In both cases, binding was dependent on Ca2+ and was totally inhibited by trifluoperazine, EGTA, and excess calmodulin. On nitrocellulose overlays, the concentration of calmodulin required to decrease binding of 125I-calmodulin by 50% was between 10(-10) and 10(-11) M. Limited proteolysis resulted in the total loss of all Ca2+-dependent binding to the 67-kDa polypeptide. Chemical cross-linking experiments identified a major calcium-dependent 125I-calmodulin:polypeptide complex in the 84-90-kDa molecular mass range and a minor complex of approximately 200 kDa. Immunoblot analysis showed that the major 67-kDa calmodulin-binding protein did not cross-react with polyclonal antibodies raised against either the calcium/calmodulin-regulated cyclic nucleotide phosphodiesterase or phosphoprotein phosphatase (calcineurin) from bovine brain.     

CENP-B is a highly conserved mammalian centromere protein with homology to the helix-loop-helix family of proteins

CENP-B is a centromere associated protein originally identified in human cells as an 80 kDa autoantigen recognized by sera from patients with anti-centromere antibodies (ACA). Recent evidence indicates that CENP-B interacts with centromeric heterochromatin in human chromosomes and may bind to a specific subset of human alphoid satellite DNA. CENP-B has not been unambiguously identified in non-primates and could, in principal, be a primate-specific alphoid DNA binding protein. In this work, a human genomic DNA segment containing the CENP-B gene was isolated and subjected to DNA sequence analysis. In vitro expression identified the site for translation initiation of CENP-B, demonstrating that it is encoded by an intronless open reading frame (ORF) in human DNA. A homologous mouse gene was also isolated and characterized. It was found to possess a high degree of homology with the human gene, containing an intronless ORF coding for a 599 residue polypeptide with 96% sequence similarity to human CENP-B. 5 and 3 flanking and untranslated sequences were conserved at a level of 94.6% and 82.7%, respectively, suggesting that the regulatory properties of CENP-B may be conserved as well. CENP-B mRNA was detected in mouse cells and tissues and an immunoreactive nuclear protein identical in size to human CENP-B was detected in mouse 3T3 cells using human ACA. Analysis of the sequence of CENP-B revealed a segment of significant similarity to a DNA binding motif identified for the helix-loop-helix (HLH) family of DNA binding proteins. These data demonstrate that CENP-B is a highly conserved mammalian protein that may be a member of the HLH protein family and suggest that it plays a role in a conserved aspect of centromere structure or function.     

Fluorescent Citrine-IgG fusion proteins produced in mammalian cells

Genetically encoded fluorescent antibodies are desirable for many applications in biotechnology, proteomics, microscopy, cell biology and molecular diagnostics, although efficient production of fluorescent IgGs in mammalian cells has been hampered by different and mutually incompatible secretion- and folding-requirements of antibodies and green fluorescent protein-derived fluorescent entities. Here, we show that this hurdle can be overcome by generating whole antibody fusions with Citrine, a modified yellow fluorescent protein that folds properly in the endoplasmic reticulum of mammalian cells. Applying optimized connector sequences, one or more Citrine molecules can be fused to different positions of IgGs without interfering with folding, secretion or function of the fusion proteins. These proteins can be transiently expressed and purified to similar yields as unmodified antibodies using standard technologies. IgG-Citrine fusions fully retain binding specificity and affinity, and can be applied to assays that require labeled IgG. A particularly interesting feature is the pH-dependency of Citrine fluorescence. This makes IgG-Citrine fusion proteins a valuable tool to track antibody target binding, internalization and subsequent intracellular trafficking to acidic compartments.     

Microtubule plus-end tracking proteins in differentiated mammalian cells

Differentiated mammalian cells are often characterized by highly specialized and polarized structure. Its formation and maintenance depends on cytoskeletal components, among which microtubules play an important role. The shape and dynamic properties of microtubule networks are controlled by multiple microtubule-associated factors. These include molecular motors and non-motor proteins, some of which accumulate specifically at the growing microtubule plus-ends (the so-called microtubule plus-end tracking proteins). Plus-end tracking proteins can contribute to the regulation of microtubule dynamics, mediate the cross-talk between microtubule ends, the actin cytoskeleton and the cell cortex, and participate in transport and positioning of structural and regulatory factors and membrane organelles. Malfunction of these proteins results in various human diseases including some forms of cancer, neurodevelopmental disorders and mental retardation. In this article we discuss recent data on microtubule dynamics and activities of microtubule plus-end binding proteins important for the physiology and pathology of differentiated mammalian cells such as neurons, polarized epithelia, muscle and sperm cells.     

Identification of two proteins associated with mammalian ATP synthase

Bovine mitochondrial ATP synthase commonly is isolated as a monomeric complex that contains 16 protein subunits and the natural IF(1) inhibitor protein in substoichiometric amounts. Alternatively ATP synthase can be isolated in dimeric and higher oligomeric states using digitonin for membrane solubilization and blue native or clear native electrophoresis for separation of the native mitochondrial complexes. Using blue native electrophoresis we could identify two ATP synthase-associated membrane proteins with masses smaller than 7 kDa and isoelectric points close to 10 that previously had been removed during purification. We show that in the mitochondrial membrane both proteins are almost quantitatively bound to ATP synthase. Both proteins had been identified earlier in a different context, but their association with ATP synthase was unknown. The first one had been named 6.8-kDa mitochondrial proteolipid because it can be isolated by chloroform/methanol extraction from mitochondrial membranes. The second one had been denoted as diabetes-associated protein in insulin-sensitive tissue (DAPIT), which may provide a clue for further functional and clinical investigations.     

Fluorescent Citrine-IgG fusion proteins produced in mammalian cells

Genetically encoded fluorescent antibodies are desirable for many applications in biotechnology, proteomics, microscopy, cell biology and molecular diagnostics, although efficient production of fluorescent IgGs in mammalian cells has been hampered by different and mutually incompatible secretion- and folding-requirements of antibodies and green fluorescent protein-derived fluorescent entities. Here, we show that this hurdle can be overcome by generating whole antibody fusions with Citrine, a modified yellow fluorescent protein that folds properly in the endoplasmic reticulum of mammalian cells. Applying optimized connector sequences, one or more Citrine molecules can be fused to different positions of IgGs without interfering with folding, secretion or function of the fusion proteins. These proteins can be transiently expressed and purified to similar yields as unmodified antibodies using standard technologies. IgG-Citrine fusions fully retain binding specificity and affinity and can be applied to assays that require labeled IgG. A particularly interesting feature is the pH-dependency of Citrine fluorescence. This makes IgG-Citrine fusion proteins a valuable tool to track antibody target binding, internalization and subsequent intracellular trafficking to acidic compartments.Key words: antibody, antibody-fusion protein, Citrine, eGFP, fluorobodies, fluorescent antibodies, FACS, immunofluorescence, DIG, IGF-1 receptor     

Posttranslational modification of proteins by isoprenoids in mammalian cells

Isoprenylation is a posttranslational modification that involves the formation of thioether bonds between cysteine and isoprenyl groups derived from pyrophosphate intermediates of the cholesterol biosynthetic pathway. Numerous isoprenylated proteins have been detected in mammalian cells. Those identified include K-, N-, and H-p21ras, ras-related GTP-binding proteins such as G25K (Gp), nuclear lamin B and prelamin A, and the gamma subunits of heterotrimeric G proteins. The modified cysteine is located in the fourth position from the carboxyl terminus in every protein where this has been studied. For p21ras, the last three amino acids are subsequently removed and the exposed cysteine is carboxylmethylated. Similar processing events may occur in lamin B and G protein gamma subunits, but the proteolytic cleavage in prelamin A occurs upstream from the modified cysteine. Lamin B and p21ras are modified by C15 farnesyl groups, whereas other proteins such as the G protein gamma subunits are modified by C20 geranylgeranyl chains. Separate enzymes may catalyze these modifications. The structural features that govern the ability of particular proteins to serve as substrates for isoprenylation by C15 or C20 groups are not completely defined, but studies of the p21ras modification using purified farnesyl:protein transferase suggest that the sequence of the carboxyl-terminal tetrapeptide is important. Isoprenylation plays a critical role in promoting the association of p21ras and the lamins with the cell membrane and nuclear envelope, respectively. Future studies of the role of isoprenylation in the localization and function of ras-related GTP-binding proteins and signal-transducing G proteins should provide valuable new insight into the link between isoprenoid biosynthesis and cell growth.     

Stable expression of Anthozoa fluorescent proteins in mammalian cells

BACKGROUND: Fluorescent proteins have become invaluable reporters in many areas of cellular and developmental biology. An enhanced version of the Aequorea victoria green fluorescent protein (AvEGFP) is the most widely used fluorescent protein. For a variety of reasons, it is useful to have alternative fluorescent proteins to AvEGFP. METHODS: The cDNA sequences for enhanced variants of the Anemonia cyan fluorescent protein (AmCyan1), as well as the Zoanthus green (ZsGreen1) and yellow (ZsYellow1) fluorescent proteins, were cloned downstream of a constitutive cytomegalovirus (CMV) promoter within a retroviral expression vector. NIH3T3, HEK293, SW620, and WM35 cells were transduced with recombinant retroviruses at a low multiplicity of infection (MOI) to bias for single-copy integration. Both unselected and stably selected cells transduced with the retroviral expression constructs were characterized. Expression of each fluorescent protein in cells was detected using flow cytometry and fluorescence microscopy with filter sets typically used for AvEGFP/fluorescein isothiocyanate (FITC) detection and was compared with the expression of AvEGFP. In addition, a fluorescence plate reader with several excitation and emission filter sets was used for detection. RESULTS: Expression of each protein was observable by fluorescence microscopy. Under given conditions of flow cytometry, the ZsGreen1 mean fluorescence was approximately 3-fold, 10-fold, and 50-fold greater than that of AvEGFP, ZsYellow1, and AmCyan1, respectively. AmCyan1, ZsGreen1, and AvEGFP were detected by a fluorescence plate reader. CONCLUSION: We determined that fluorescent proteins from Anthozoa species are detectable using a standard flow cytometer and fluorescence microscope. All of the mammalian cell lines tested expressed detectable levels of fluorescent proteins from stable integrated provirus. In cell lines where the AvEGFP protein is toxic or poorly expressed, these Anthozoa fluorescent proteins may serve as alternative fluorescent reporters.     

Migration of centromere proteins in rabbit spermatids.

Human autoantibodies were used to localize centromere proteins by immunoelectron microscopy, immunofluorescence, and confocal microscopy in isolated cells and in cryosections of rabbit testis. A computer-assisted three-dimensional reconstruction of the positions and sizes of fluorescent spots allowed us to follow their movements during the different phases of spermiogenesis. In very young spermatids, the centromeres were distributed within a space separated from both the external nuclear limits and the nuclear core. They moved towards the nuclear center in cap phase spermatids, where they clustered into a few large centromeric masses. In preelongating spermatids, the immunolabeled proteins were dispersed within an equatorial area, where they formed one large mass. In late spermatids, the mass moved towards the posterior part of the nucleus, and, in the spermatozoon, the two basal knobs located at the base of the nuclei were the only strongly immunolabeled structures, while no labeling of the main part of the nucleus was observed. Since the number of centromeres remained close to the number of chromosomes until the cap phase of spermatid differentiation, we hypothesize that the labeling of young spermatids corresponds to centrometric proteins associated with their specific DNA counterparts, while the centromere proteins, possibly detached from their DNA loci, were released from nuclei of old spermatids in the same way as are histones and transition proteins.     

Identification of a family of human centromere proteins using autoimmune sera from patients with scleroderma

We have examined preimmune serum samples from a patient who progressively developed the symptoms of scleroderma CREST over a period of several years. During this period, anti-centromere antibodies (recognized by indirect immunofluorescence) appeared in the serum. Concomitant with the appearance of the anti-centromere antibodies, antibody species recognizing three chromosomal antigens in immunoblots of SDS polyacrylamide gels appeared in the patient's serum. These antigens migrate with electrophoretic mobilities corresponding to M_r=17, 80, and 140 kilodaltons (kd). Affinity-eluted antibody fractions recognizing the antigens have been prepared from sera of three other patients. Indirect immunofluorescence labeling of mitotic cells using these antibody fractions demonstrates that the antigens are centromere components. We designate them CENP (CENtromere Protein) — A (17kd), CENP-B (80kd), and CENP-C (140kd). The three CENP antigens share antigenic determinants. Immunoblotting experiments show that these patients make antibody species recognizing at least three distinct epitopes on CENP-B and two on CENP-C. Sera from different patients contain different mixtures of the antibody species.     

The nonhistone proteins of developing mammalian erythroid cells

Anaemic rabbit bone marrow cells, labelled with [35S]methionine, were separated at unit gravity. Chromatin was isolated from these cells and proteins were separated from DNA, using urea/salt extraction. Two-dimensional gel electrophoresis of the nonhistone proteins showed that these proteins appeared to change quantitatively but not qualitatively, with one important exception, as cell development proceeded. The one protein that did change had a molecular weight of approximately 20,000 and was very basic. This protein was synthesised at low levels in the early cells, but its synthesis was seen to increase at the polychromatic stage of cell development, just prior to nuclear condensation. Treatment of bone marrow cells with sodium butyrate was shown to increase the synthesis of this protein in the early cells.