Cytoplasm Of HeLa Cells Research Articles

A protein secreted by the respiratory pathogenChlamydia pneumoniaeimpairs IL-17 signalling via interaction with human Act1

Chlamydia pneumoniae is a common respiratory pathogen that has been associated with a variety of chronic diseases including asthma and atherosclerosis. Chlamydiae are obligate intracellular parasites that primarily infect epithelial cells where they develop within a membrane-bound vacuole, termed an inclusion. Interactions between the microorganism and eukaryotic cell can be mediated by chlamydial proteins inserted into the inclusion membrane. We describe here a novel C. pneumoniae-specific inclusion membrane protein (Inc) CP0236, which contains domains exposed to the host cytoplasm. We demonstrate that, in a yeast two-hybrid screen, CP0236 interacts with the NFκB activator 1 (Act1) and this interaction was confirmed in HeLa 229 cells where ectopically expressed CP0236 was co-immunoprecipitated with endogenous Act1. Furthermore, we demonstrate that Act1 displays an altered distribution in the cytoplasm of HeLa cells infected with C. pneumoniae where it associates with the chlamydial inclusion membrane. This sequestration of Act1 by chlamydiae inhibited recruitment of the protein to the interleukin-17 (IL-17) receptor upon stimulation of C. pneumoniae-infected cells with IL-17A. Such inhibition of the IL-17 signalling pathway led to protection of Chlamydia-infected cells from NFκB activation in IL-17-stimulated cells. We describe here a unique strategy employed by C. pneumoniae to achieve inhibition of NFκB activation via interaction of CP0236 with mammalian Act1.

The role of hydrophobic amino acid grafts in the enhancement of membrane-disruptive activity of pH-responsive pseudo-peptides

pH-responsive polymers have been synthesised by grafting l-valine (PV-75), l-leucine (PL-75) and l-phenylalanine (PP-75) onto the pendant carboxylic acid moieties of a pseudo-peptide, poly( l-lysine iso-phthalamide), at a stoichiometric degree of substitution of 75 mol%. The effect of such modification on the pH-, concentration- and time-dependent cell membrane-disruptive activity of the grafted polymers has been investigated using a haemolysis model. At 0.025 mg mL −1, the grafted polymers were almost non-haemolytic at pH 7.4, but mediated considerable membrane lysis after 60 min in the pH range characteristic of early endosomes, which ranked in the order: PP-75 > PL-75 > PV-75 > poly( l-lysine iso-phthalamide). PP-75 was 35-fold more lytic on a molar basis than the membrane-lytic peptide melittin. With increasing concentration, the grafted polymers showed an increased ability to lyse cell membranes and caused noticeable membrane disruption at physiological pH. The mechanism of the polymer-mediated membrane destabilisation has been investigated. The in-vitro cytotoxicity of the grafted polymers has been assessed using a propidium iodide fluorescence assay. It has been demonstrated by confocal microscopy that the grafted polymers can induce a significant release of endocytosed materials into the cytoplasm of HeLa cells, which is a feature critical for drug delivery applications.

RXFP1-inactive relaxin activates human glucocorticoid receptor: Further investigations into the relaxin–GR pathway

The relaxin peptide family regulates diverse biological functions (central nervous processes, reproduction, cardiovascular and kidney function, and connective tissue composition) through different G protein-coupled receptors. We reported earlier that human relaxin-2 and porcine relaxin additionally interact with the human glucocorticoid receptor (GR) in an agonistic manner. Here we investigated whether the membrane receptor RXFP1 is critically involved in this pathway. We used chemically modified porcine relaxin which was biologically inactive at RXFP1. Native porcine relaxin, but not the modified peptide affected RXFP1-dependent and GR-independent readouts: ERK-1/2 and Akt phosphorylation as well as up-regulation of Akt and endothelin type-B receptor. In contrast, relaxin and modified relaxin inhibited endotoxin-stimulated secretion of TNF-α and IL-6 by human macrophages, an effect sensitive to the glucocorticoid receptor antagonists RU-486 and D-06. Both relaxins caused Ser 211 phosphorylation of GR, a biomarker of agonist-related receptor activation. Relaxin-induced accumulation of Ser 211-phosphorylated GR was found in the cytoplasm and nucleus of HeLa cells, in endothelial cells, and in transfected HT-29 cells. In AP-1-luciferase assays, relaxin and modified relaxin inhibited endotoxin-induced activation of AP-1, a transcription factor essentially involved in endotoxin signaling. This study suggests that the inability of relaxin to interact with its membrane receptor does not interfere with its ability to activate GR.

Synthesis of an AIE-active fluorogen and its application in cell imaging

A fluorogen named 1-decyl-1-methyl-2,5-bis{4-[(N,N-diethylamino)methyl]phenyl}-3,4-diphenylsilole (3) was synthesized. It emitted weakly as isolated molecule but strongly as supramolecular aggregate, showing a characteristic behavior of aggregation-induced emission (AIE). The molecules of 3 formed highly emissive nanoparticles in aqueous media, which quickly and selectively marked cytoplasm of HeLa cells and posed no toxicity to the living cells. The fluorogen is thus a promising candidate mate-rial for cell imaging as a sensitive, selective and cytocompatible biosensor.

Aqueous solution behaviour and membrane disruptive activity of pH-responsive PEGylated pseudo-peptides and their intracellular distribution

The effect of PEGylation on the aqueous solution properties and cell membrane disruptive activity of a pH-responsive pseudo-peptide, poly( l-lysine iso-phthalamide), has been investigated by dynamic light scattering, haemolysis and lactate dehydrogenase (LDH) assays. Intracellular trafficking of the polymers has been examined using confocal and fluorescence microscopy. With increasing degree of PEGylation, the modified polymers can form stabilised compact structures with reduced mean hydrodynamic diameters. Poly( l-lysine iso-phthalamide) with a low degree of PEGylation (17.4 wt%) retained pH-dependent solution behaviour and showed enhanced kinetic membrane disruptive activity compared to the parent polymer. It facilitated trafficking of endocytosed materials into the cytoplasm of HeLa cells. At levels of PEGylation in excess of 25.6 wt%, the modified polymers displayed a single particle size distribution unresponsive to pH, as well as a decrease in cell membrane lytic ability. The mechanism involved in membrane destabilisation was also investigated, and the potential applications of these modified polymers in drug delivery were discussed.

Generation and Characterization of a Monoclonal Antibody Against NPI-1 Subfamily of Importinα

Most karyophilic proteins are transported into the nucleus through the importin-mediated pathway. Importin alpha acts as a receptor for classical nuclear localization signal (NLS)-containing proteins. At present, the existence of several isoforms of importin alpha in mammals is known. In this study we report on the generation of a rat monoclonal antibody (MAb) 2D9 to importin alpha NPI-1 subfamily members (importin alpha5/NPI-1 and importin alpha7/S2) using the rat lymph node method and the characterization of this antibody. In several different cultured cell extracts, MAb 2D9 reacted to endogenous NPI-1 subfamily in Western blotting experiments. Epitope mapping using recombinant deletion mutants indicated that MAb 2D9 recognized arm motif in importin alpha5/NPI-1. Using immunofluorescence, MAb 2D9 detected NPI-1 subfamily in the cytoplasm of HeLa cells. Moreover, endogenous NPI-1 subfamily was dominantly localized in the nuclei of H(2)O(2)-treated HeLa cells, suggesting that NPI-1 subfamily accumulates in the nucleus in response to oxidative stress, like importin alpha1/Rch1.

Ubiquitin Chain Editing Revealed by Polyubiquitin Linkage-Specific Antibodies

Posttranslational modification of proteins with polyubiquitin occurs in diverse signaling pathways and is tightly regulated to ensure cellular homeostasis. Studies employing ubiquitin mutants suggest that the fate of polyubiquitinated proteins is determined by which lysine within ubiquitin is linked to the C terminus of an adjacent ubiquitin. We have developed linkage-specific antibodies that recognize polyubiquitin chains joined through lysine 63 (K63) or 48 (K48). A cocrystal structure of an anti-K63 linkage Fab bound to K63-linked diubiquitin provides insight into the molecular basis for specificity. We use these antibodies to demonstrate that RIP1, which is essential for tumor necrosis factor-induced NF-kappaB activation, and IRAK1, which participates in signaling by interleukin-1beta and Toll-like receptors, both undergo polyubiquitin editing in stimulated cells. Both kinase adaptors initially acquire K63-linked polyubiquitin, while at later times K48-linked polyubiquitin targets them for proteasomal degradation. Polyubiquitin editing may therefore be a general mechanism for attenuating innate immune signaling.

Construction of eukaryotic expression vector encoding ATP synthase lipid-binding protein-like protein gene of Sj and its expression in HeLa cells

Objective To clone and construct the recombinant plasmid containing ATP synthase lipid-binding protein-like protein gene of Schistosoma japonicum,(SjAslp) and transfer it into mammalian cells to express the objective protein. Methods By polymerase chain reaction (PCR) technique, SjAslp was amplified from the constructed recombinant plasmid pBCSK +/SjAslp, and inserted into cloning vector pUCm-T. Then, SjAslp was subcloned into an eukaryotic expression vector pcDNA3.1(+). After identifying it by PCR, restrictive enzymes digestion and DNA sequencing, the recombinant plasmid was transfected into HeLa cells using electroporation, and the expression of the recombinant protein was analyzed by immunocytochemical assay. Results The specific gene fragment of 558 bp was successfully amplified. The DNA vaccine of SjAslp was successfully constructed. Immunocytochemical assay showed that SjAslp was expressed in the cytoplasm of HeLa cells. Conclusion SjAslp gene can be expressed in eukaryotic system, which lays the foundation for development of the SjAslp DNA vaccine against schitosomiasis.

Monodisperse Magnetite Nanoparticles Coupled with Nuclear Localization Signal Peptide for Cell‐Nucleus Targeting

Functionalization of monodisperse superparamagnetic magnetite (Fe(3)O(4)) nanoparticles for cell specific targeting is crucial for cancer diagnostics and therapeutics. Targeted magnetic nanoparticles can be used to enhance the tissue contrast in magnetic resonance imaging (MRI), to improve the efficiency in anticancer drug delivery, and to eliminate tumor cells by magnetic fluid hyperthermia. Herein we report the nucleus-targeting Fe(3)O(4) nanoparticles functionalized with protein and nuclear localization signal (NLS) peptide. These NLS-coated nanoparticles were introduced into the HeLa cell cytoplasm and nucleus, where the particles were monodispersed and non-aggregated. The success of labeling was examined and identified by fluorescence microscopy and MRI. The work demonstrates that monodisperse magnetic nanoparticles can be readily functionalized and stabilized for potential diagnostic and therapeutic applications.

A Versatile Ditopic Ligand System for Sensitizing the Luminescence of Bimetallic Lanthanide Bio‐Imaging Probes

The homoditopic ligand 6,6'-[methylenebis(1-methyl-1H-benzimidazole-5,2-diyl)]bis(4-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}pyridine-2-carboxylic acid) (H(2)L(C2)) has been tailored to self-assemble with lanthanide ions (Ln(III)), which results in the formation of neutral bimetallic helicates with the overall composition [Ln(2)(L(C2))(3)] and also provides a versatile platform for further derivatization. The grafting of poly(oxyethylene) groups onto the pyridine units ensures water solubility, while maintaining sizeable thermodynamic stability and adequate antenna effects for the excitation of both visible- and NIR-emitting Ln(III) ions. The conditional stability constants (log beta(23)) are close to 25 at physiological pH and under stoichiometric conditions. The ligand triplet state features adequate energy (0-phonon transition at approximately 21 900 cm(-1)) to sensitize the luminescence of Eu(III) (Q=21 %) and Tb(III) (11 %) in aerated water at pH 7.4. The emission of several other VIS- and NIR-emitting ions, such as Sm(III) (Q=0.38 %) or Yb(III) (0.15 %), for which in cellulo luminescence is evidenced for the first time, is also sensitized. The Eu(III) emission spectrum arises from a main species with pseudo-D(3) symmetry and without coordinated water. The cell viability of several cancerous cell lines (MCF-7, HeLa, Jurkat and 5D10) is unaffected if incubated with up to 500 microM [Eu(2)(L(C2))(3)] during 24 h. Bright Eu(III) emission is seen for incubation concentrations above 10 microM and after a 15-minute loading time; similar images are obtained with Tb(III) and Sm(III). The helicates probably permeate into the cytoplasm of HeLa cells by endocytosis. The described luminescent helical stains are robust chemical species which remain undissociated in the cell medium and in presence of other complexing agents, such as edta, dtpa, citrate or L-ascorbate. Their derivatization, which would open the way to the sensing of targeted in cellulo phenomena, is currently under investigation.

Precise Measurement of Diffusion Coefficients using Scanning Fluorescence Correlation Spectroscopy

We have implemented scanning fluorescence correlation spectroscopy (sFCS) for precise determination of diffusion coefficients of fluorescent molecules in solution. The measurement volume where the molecules are excited, and from which the fluorescence is detected, was scanned in a circle with radius comparable to its size at frequencies 0.5–2 kHz. The scan radius R, determined with high accuracy by careful calibration, provides the spatial measure required for the determination of the diffusion coefficient D, without the need to know the exact size of the measurement volume. The difficulties in the determination of the measurement volume size have limited the application of standard FCS with fixed measurement volume to relative measurements, where the diffusion coefficient is determined by comparison with a standard. We demonstrate, on examples of several common fluorescent dyes, that sFCS can be used to measure D with high precision without a need for a standard. The correct value of D can be determined in the presence of weak photobleaching, and when the measurement volume size is modified, indicating the robustness of the method. The applicability of the presented implementation of sFCS to biological systems in demonstrated on the measurement of the diffusion coefficient of eGFP in the cytoplasm of HeLa cells. With the help of simulations, we find the optimal value of the scan radius R for the experiment.

Interaction between Parasitophorous Vacuolar Membrane-associated GRA3 and Calcium Modulating Ligand of Host Cell Endoplasmic Reticulum in the Parasitism of Toxoplasma gondii

A monoclonal antibody against Toxoplasma gondii of Tg556 clone (Tg556) blotted a 29 kDa protein, which was localized in the dense granules of tachyzoites and secreted into the parasitophorous vacuolar membrane (PVM) after infection to host cells. A cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg556, and the full-length was completed by 5'-RACE of 2,086 bp containing an open reading frame (ORF) of 669 bp. The ORF encoded a polypeptide of 222 amino acids homologous to the revised GRA3 but not to the first reported one. The polypeptide has 3 hydrophobic moieties of an N-terminal stop transfer sequence and 2 transmembrane domains (TMD) in posterior half of the sequence, a cytoplasmic localization motif after the second TMD and an endoplasmic reticulum (ER) retrival motif in the C-terminal end, which suggests GRA3 as a type III transmembrane protein. With the ORF of GRA3, yeast two-hybrid assay was performed in HeLa cDNA expression library, which resulted in the interaction of GRA3 with calcium modulating ligand (CAMLG), a type II transmembrane protein of ER. The specific binding of GRA3 and CAMLG was confirmed by glutathione S-transferase (GST) pull-down and immunoprecipitation assays. The localities of fluorescence transfectionally expressed from GRA3 and CAMLG plasmids were overlapped completely in HeLa cell cytoplasm. In immunofluorescence assay, GRA3 and CAMLG were shown to be co-localized in the PVM of host cells. Structural binding of PVM-inserted GRA3 to CAMLG of ER suggested the receptor-ligand of ER recruitment to PVM during the parasitism of T. gondii.

Cytoplasm‐localized SIRT1 enhances apoptosis

In general, SIRT1 is localized in nuclei. Here, we showed that endogenous and exogenous SIRT1 were both able to partially localize in cytoplasm in certain cell lines, and cytoplasm-localized SIRT1 was associated with apoptosis and led to increased sensitivity to apoptosis. Furthermore, we demonstrated that translocation of nucleus-localized SIRT1 from nuclei to cytoplasm was the main pathway leading to localization of SIRT1 in cytoplasm. In HeLa cells, wild type SIRT1 was completely localized in nuclei. By truncation of two predicted nuclear localization signals or fusion with an exogenous nuclear export signal, SIRT1 was partially localized in cytoplasm of HeLa cells and resulted in increased sensitivity to apoptosis. The apoptosis enhanced by cytoplasm-localized SIRT1 was independent of its deacetylase activity, but dependent on caspases. SIRT1 was distributed in cytoplasm at metaphase during mitosis, and overexpression of SIRT1 significantly augmented apoptosis for cells at metaphase. In summary, we found SIRT1 is able to localize in cytoplasm, and cytoplasm-localized SIRT1 enhances apoptosis.

Nuclear and cytoplasmic peroxiredoxin-1 differentially regulate NF-κB activities

Peroxiredoxins (Prx) are widely distributed and abundant proteins, which control peroxide concentrations and related signaling mechanisms. Prx1 is found in the cytoplasm and nucleus, but little is known about compartmentalized Prx1 function during redox signaling and oxidative stress. We targeted expression vectors to increase Prx1 in nuclei (NLS-Prx1) and cytoplasm (NES-Prx1) in HeLa cells. Results showed that NES-Prx1 inhibited NF-κB activation and nuclear translocation. In contrast, increased NLS-Prx1 did not affect NF-κB nuclear translocation but increased activity of a NF-κB reporter. Both NLS-Prx1 and NES-Prx1 inhibited NF-κB p50 oxidation, suggesting that oxidation of the redox-sensitive cysteine in p50's DNA-binding domain is regulated via peroxide metabolism in both compartments. Interestingly, following treatment with H 2O 2, nuclear thioredoxin-1 (Trx1) redox status was protected by NLS-Prx1, and cytoplasmic Trx1 was protected by NES-Prx1. Compartmental differences from increasing Prx1 show that the redox poise of cytoplasmic and nuclear thiol systems can be dynamically controlled through peroxide elimination. Such spatial resolution and protein-specific redox differences imply that the balance of peroxide generation/metabolism in microcompartments provides an important specific component of redox signaling.

Conjugation of a New Two-Photon Fluorophore to Poly(ethylenimine) for Gene Delivery Imaging

We report herein the molecular engineering of an efficient two-photon absorbing (TPA) chromophore based on a donor-donor bis-stilbenyl entity to allow conjugation with biologically relevant molecules. The dye has been functionalized using an isothiocyanate moiety to conjugate it with the amine functions of poly(ethylenimine) (PEI), which is a cationic polymer commonly used for nonviral gene delivery. Upon conjugation, the basic architecture and photophysical properties of the active TPA chromophore remain unchanged. At the usual N/P ratio (ratio of the PEI positive charges to the DNA negative charges) of 10 used for transfection, the transfection efficiency and cytotoxicity of the labeled PEI/DNA complexes were found to be comparable to those of the unlabeled PEI/DNA complexes. Moreover, when used in combination with unlabeled PEI (at a ratio of 1 labeled PEI to 3 unlabeled PEI), the labeled PEI does not affect the size of the complexes with DNA. The labeled PEI was successfully used in two-photon fluorescence correlation spectroscopy measurements, showing that at N/P = 10 most PEI molecules are free and the diffusion coefficient of the complexes is consistent with the 360 nm size measured by quasielastic light scattering. Finally, two-photon images of the labeled PEI/DNA complexes confirmed that the complexes enter into the cytoplasm of HeLa cells by endocytosis and hardly escape from the endosomes. As a consequence, the functionalized TPA chromophore appears to be an adequate tool to label the numerous polyamines used in nonviral gene delivery and characterize their complexes with DNA in two-photon applications.

Synthesis, Stability, and Cellular Internalization of Gold Nanoparticles Containing Mixed Peptide−Poly(ethylene glycol) Monolayers

Gold nanoparticles have shown great promise as therapeutics, therapeutic delivery vectors, and intracellular imaging agents. For many biomedical applications, selective cell and nuclear targeting are desirable, and these remain a significant practical challenge in the use of nanoparticles in vivo. This challenge is being addressed by the incorporation of cell-targeting peptides or antibodies onto the nanoparticle surface, modifications that frequently compromise nanoparticle stability in high ionic strength biological media. We describe herein the assembly of poly(ethylene glycol) (PEG) and mixed peptide/PEG monolayers on gold nanoparticle surfaces. The stability of the resulting bioconjugates in high ionic strength media was characterized as a function of nanoparticle size, PEG length, and monolayer composition. In total, three different thiol-modified PEGs (average molecular weight (MW), 900, 1500, and 5000 g mol-1), four particle diameters (10, 20, 30, and 60 nm), and two cell-targeting peptides were explored. We found that nanoparticle stability increased with increasing PEG length, decreasing nanoparticle diameter, and increasing PEG mole fraction. The order of assembly also played a role in nanoparticle stability. Mixed monolayers prepared via the sequential addition of PEG followed by peptide were more stable than particles prepared via simultaneous co-adsorption. Finally, the ability of nanoparticles modified with mixed PEG/RME (RME = receptor-mediated endocytosis) peptide monolayers to target the cytoplasm of HeLa cells was quantified using inductively coupled plasma optical emission spectrometry (ICP-OES). Although it was anticipated that the MW 5000 g mol-1 PEG would sterically block peptides from access to the cell membrane compared to the MW 900 PEG, nanoparticles modified with mixed peptide/PEG 5000 monolayers were internalized as efficiently as nanoparticles containing mixed peptide/PEG 900 monolayers. These studies can provide useful cues in the assembly of stable peptide/gold nanoparticle bioconjugates capable of being internalized into cells.

Characterization and Identification of a Steroid Receptor-Binding Protein, SRB-RGS

We cloned the cDNA of a novel steroid receptor-binding protein, SRB-RGS, which suppressed the estrogen receptor (ER)alpha-mediated and other promoter-driven transcriptional activities. This study revealed the interaction between the full-length SRB-RGS and full-length ERalpha or ERbeta by a coimmunoprecipitation assay. The full-length SRB-RGS and full-length ERalpha interacted in COS-7 cell by a mammalian two-hybrid system. The interaction between intrinsic SRB-RGS and ERs in the nuclear ER extract from the rat uteri was observed by the gel-shift assay. These results strongly suggested that SRB-RGS interacts with ERs bound to DNA (estrogen response element) in the nuclei of the cells. SRB-RGS suppressed very efficiently the ERalpha-, ERbeta-, and ERalpha+ERbeta-mediated transcriptional activities. Green fluorescence of enhanced green fluorescence protein (EGFP)-tagged SRB-RGS was localized both in the nucleus and in the cytoplasm. Intrinsic SRB-RGS was immunostained in the nucleus and the cytoplasm of HeLa cells. The putative SRB-RGS deduced from cDNA sequence was identified by the immunostaining and Western blotting by using the anti-SRB-RGS antibody. Overexpression of SRB-RGS induced the cell death in the HeLa cells. The nucleotide sequence of SRB-RGS cDNA that we cloned previously is identical with that of the newly isolated RGS3 cDNA. SRB-RGS could interact with ERs bound DNA in the nuclei of the cells and suppressed the ERs-mediated transcriptional activities.

Synthesis and cellular uptake of cell delivering 2,6-pyridinediylbisalkanamide submicron-sized sheets in HeLa cells

2,6-Pyridinediylbisalkanamides were synthesized, using diaminopyridine (DAP) as a linker and alkyl chains of varying lengths, that upon self-assembly form submicron-sized sheets and their uptake into the cytoplasm of HeLa cells was studied by confocal microscopy.

A Monoclonal Antibody against Human Homocysteine-Induced Endoplasmic Reticulum Protein (Herp): A Useful Tool for Evaluating Endoplasmic Reticulum Stress

Hyperhomocysteinemia has been reported as one of the risk factors for vascular damage. Homocysteine induces endoplasmic reticulum (ER) stress in vascular endothelial cells, which is followed by production of homocysteine-induced ER protein (Herp). Herp has been thought to have a protective role against ER stress and inhibition of apoptosis, but the details are still obscure. To detect Herp protein precisely, we established a murine hybridoma clone producing an anti-human Herp monoclonal antibody (mAb), named HT2. The specific binding of HT2 mAb to Herp was confirmed by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. In ELISA, HT2 mAb was able to bind to Herp in a dose-dependent manner, and its binding was interrupted by recombinant Herp. In Western blot analysis, a 54-kDa band corresponding to Herp was detected with HT2 mAb in the membrane fraction of untreated HeLa cells, and its expression was remarkably increased in ER-stressed HeLa cells that had been treated with homocysteine, thapsigargin, or 2-mercaptoethanol. Importantly, the signal was eliminated by absorption of HT2 mAb with recombinant Herp prior to incubation with the blotted membrane. Immunofluorescence microscopy revealed that HT2 mAb stained the perinuclear cytoplasm of ER-stressed HeLa cells, which was similar to the staining pattern with anti-KDEL (Lys-Asp-Glu-Leu) mAb that recognizes the ER. In contrast, untreated HeLa cells were weakly stained with HT2 mAb. Thus, the HT2 mAb is useful in the quantitative and/or qualitative detection of Herp and to study the role of Herp at a variety of pathological states.

Male Germ Cell Expression of the PAS Domain Kinase PASKIN and its Novel Target Eukaryotic Translation Elongation Factor eEF1A1

PASKIN links energy flux and protein synthesis in yeast, regulates glycogen synthesis in mammals, and has been implicated in glucose-stimulated insulin production in pancreatic β-cells. Using newly generated monoclonal antibodies, PASKIN was localized in the nuclei of human testis germ cells and in the midpiece of human sperm tails. A speckle-like nuclear pattern was observed for endogenous PASKIN in HeLa cells in addition to its cytoplasmic localization. By yeast two-hybrid screening, we identified the multifunctional eukaryotic translation elongation factor eEF1A1 as a novel interaction partner of PASKIN. This interaction was mapped to the PAS A and kinase domains of PASKIN and to the C-terminus of eEF1A1 using mammalian two-hybrid and GST pull-down assays. Kinase assays, mass spectrometry and site-directed mutagenesis revealed PASKIN auto-phosphorylation as well as eEF1A1 target phosphorylation mainly but not exclusively at Thr432. Wild-type but not kinase-inactive PASKIN increased the in vitro translation of a reporter cRNA. Whereas eEF1A1 did not localize to the nucleus, it co-localizes with PASKIN to the cytoplasm of HeLa cells. The two proteins also showed a remarkably similar localization in the midpiece of the sperm tail. These data suggest regulation of eEF1A1 by PASKIN-dependent phosphorylation in somatic as well as in sperm cells.