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PubMed2004

A methylation-mediator complex in hormone signaling.

The recruitment of coactivators by nuclear hormone receptors (NRs) promotes transcription by subverting chromatin-mediated repression. Although the histone methylation enzyme CARM1 and an ATP-remodeling complex have been individually implicated in nuclear receptor-dependent transcription, neither a functional nor mechanistic linkage between these systems has been identified. In the process of purifying endogenous CARM1-interacting proteins, we identified an associated complex, nucleosomal methylation activator complex (NUMAC), which includes at least eight components of SWI/SNF, including the ATPase BRG1. In the NUMAC complex, the methylase, CARM1, acquires the ability to covalently modify nucleosomal histones, and the directed nucleosome versus free core histone methylation-specificity change is increased dramatically. Reciprocally, CARM1 stimulates the ATPase activity of BRG1, a key component in nucleosome remodeling. In vivo, CARM1 and BRG1 coassemble on an estrogen receptor (ER)-target gene to cooperatively activate ER-dependent transcription. This association of ATP-remodeling factors with HMT CARM1 defines a new component of regulation in the nuclear hormone-signaling pathway.

Adenosine Triphosphate↗
PubMed1999

An inactive open complex mediated by an UP element at Escherichia coli promoters.

A specific interaction between the alpha subunit of RNA polymerase and an A+T-rich upstream sequence (UP element) stimulates transcription at some promoters in Escherichia coli. We found that RNA polymerase formed a heparin-resistant nonproductive initiation complex at the malT promoter which has an A+T-rich upstream sequence that begins 9 bp upstream of the -35 region. Substitution of other sequences for the A+T-rich sequence eliminated both the formation of heparin-resistant complexes and alpha binding to the malT promoter. A 5-bp deletion between the A+T-rich sequence and the -35 region increased promoter activity. The UP element derived from the rrnB P1 promoter stimulated transcription of the malT core promoter when placed 4 bp upstream from the malT -35 region, but insertion of an additional 4 bp between the rrnB P1 UP element and the -35 element eliminated transcription activity without eliminating heparin-resistant complex formation. Similar UP element effects were observed in hybrids with the lac core promoter, even though the region around the transcription start site was melted in both productive and nonproductive complexes. We conclude that UP elements can mediate the formation of both productive and nonproductive open complexes, depending on their location with respect to the core promoter.

Base Sequence↗
PubMed2001

Pivotal role of VASP in Arp2/3 complex-mediated actin nucleation, actin branch-formation, and Listeria monocytogenes motility.

The Listeria monocytogenes ActA protein mediates actin-based motility by recruiting and stimulating the Arp2/3 complex. In vitro, the actin monomer-binding region of ActA is critical for stimulating Arp2/3-dependent actin nucleation; however, this region is dispensable for actin-based motility in cells. Here, we provide genetic and biochemical evidence that vasodilator-stimulated phosphoprotein (VASP) recruitment by ActA can bypass defects in actin monomer-binding. Furthermore, purified VASP enhances the actin-nucleating activity of wild-type ActA and the Arp2/3 complex while also reducing the frequency of actin branch formation. These data suggest that ActA stimulates the Arp2/3 complex by both VASP-dependent and -independent mechanisms that generate distinct populations of actin filaments in the comet tails of L. monocytogenes. The ability of VASP to contribute to actin filament nucleation and to regulate actin filament architecture highlights the central role of VASP in actin-based motility.

Actin-Related Protein 2↗
PubMed1988

Accelerated electron transfer between metal complexes mediated by DNA.

DNA-mediated long-range electron transfer from photoexcited 1,10-phenanthroline complexes of ruthenium, Ru(phen)2(3)+, to isostructural complexes of cobalt(III), rhodium(III), and chromium(III) bound along the helical strand. The efficiency of transfer depended upon binding mode and driving force. For a given donor-acceptor pair, surface-bound complexes showed greater rate enhancements than those that were intercalatively bound. Even in rigid glycerol at 253 K, the rates for donor-acceptor pairs bound to DNA remained enhanced. For the series of acceptors, the greatest enhancement in electron-transfer rate was found with chromium, the acceptor of intermediate driving force. The DNA polymer appears to provide an efficient intervening medium to couple donor and acceptor metal complexes for electron transfer.

Binding, Competitive↗
PubMed2000

Evidence for major histocompatibility complex-mediated effects on spermatogenesis in humans.

Studies in fertile and infertile populations suggest an influence of genes of the major histocompatibility complex (MHC) on reproduction, although it remains unresolved if MHC-mediated effects on fertility are based on direct immunological or non-immunological effects of human leukocyte antigen (HLA) genes or rather on defects in MHC-associated non-HLA genes which affect gamete quality and embryonic development. We analysed allele frequencies for HLA class II loci DQA1, DQB1 and DRB1 and HLA class II haplotype frequencies in couples with tubal and andrological infertility who were treated with assisted reproductive techniques. Males with severe andrological infertility had significantly different allele frequencies for all three HLA loci when compared to males with normozoospermia, whereas no difference was found in the females. Differences in allele frequencies were stronger when only males whose partners achieved pregnancies after assisted reproduction treatment were compared. In those subgroups, we could also observe significant differences in three locus HLA class II haplotype frequencies. In summary, patients with male factor infertility differ in their HLA class II allele constitution from males with normozoospermia, which suggests that genes identical to or located in close vicinity to HLA class II genes may influence spermatogenesis and male gamete function.

Alleles↗
PubMed2004

The chromatin-remodeling BAF complex mediates cellular antiviral activities by promoter priming.

The elicitation of cellular antiviral activities is dependent on the rapid transcriptional activation of interferon (IFN) target genes. It is not clear how the interferon target promoters, which are organized into chromatin structures in cells, rapidly respond to interferon or viral stimulation. In this report, we show that alpha IFN (IFN-alpha) treatment of HeLa cells induced hundreds of genes. The induction of the majority of these genes was inhibited when one critical subunit of the chromatin-remodeling SWI/SNF-like BAF complexes, BAF47, was knocked down via RNA interference. Inhibition of BAF47 blocked the cellular response to viral infection and impaired cellular antiviral activity by inhibiting many IFN- and virus-inducible genes. We show that the BAF complex was required to mediate both the basal-level expression and the rapid induction of the antiviral genes. Further analyses indicated that the BAF complex primed some IFN target promoters by utilizing ATP-derived energy to maintain the chromatin in a constitutively open conformation, allowing faster and more potent induction after IFN-alpha treatment. We propose that constitutive binding of the BAF complex is an important mechanism for the IFN-inducible promoters to respond rapidly to IFN and virus stimulation.

Antigens, Differentiation↗
PubMed2005

TNF regulates leukocyte-endothelial cell interactions and microvascular dysfunction during immune complex-mediated inflammation.

1. The aim of this study was to assess directly the role of TNF in immune complex-induced leukocyte-endothelial cell interactions and microvascular dysfunction. 2. Intravital microscopy was used to examine immune complex-induced leukocyte rolling, adhesion and emigration and microvascular permeability in cremasteric postcapillary venules in wild-type and TNF(-/-) mice. The reverse passive Arthus (RPA) reaction was used to localize immune complex formation to the cremaster muscle. 3. In wild-type mice, immune complex deposition induced a reduction in leukocyte rolling velocity and increases in leukocyte adhesion and emigration. In TNF(-/-) mice, the immune complex-induced reduction in leukocyte rolling velocity was significantly attenuated, and leukocyte adhesion and emigration were also significantly reduced relative to responses in wild-type mice. 4. The alterations in TNF(-/-) mice were associated with decreased expression of endothelial P-selectin and VCAM-1, and an absence of E-selectin-dependent rolling normally seen in wild-type mice at the peak of the response. In addition, the level of immune complex-induced microvascular permeability was attenuated in TNF(-/-) mice. 5. These findings demonstrate that in immune complex-induced inflammation, TNF promotes leukocyte rolling and adhesive interactions, and entry of leukocytes into sites of immune complex deposition, in part via the increased expression and/or function of endothelial P-selectin, E-selectin and VCAM-1. In addition, this increase in leukocyte recruitment mediated by TNF correlates directly with an increase in microvascular injury.

Animals↗
PubMed1983

Evidence supporting a role for immune complex-mediated inflammation in the pathogenesis of bullous lesions of systemic lupus erythematosus.

Evidence supporting an immune complex pathogenesis of bullous lesions in systemic lupus erythematosus includes immune deposits, acute inflammation, and blister formation at the cutaneous basement membrane zone. Since cutaneous immune deposits are a general feature of lupus, an attempt has been made to determine whether deposits in lupus patients with blisters are functionally different from those in patients without blisters. Skin was obtained from 4 consecutive patients with blisters and 14 controls. The groups were matched for clinical and serologic features, duration and activity of disease, and treatment. Skin was examined by direct immunofluorescence for immune deposits and by the leukocyte attachment assay for quantification of complement-activating immune complexes. Clinically normal, viable skin from 1 patient with blisters and 1 patient without blisters was incubated in organ culture with normal human leukocytes and serum complement. All patients in both groups had immune deposits at the basement membrane zone with an equivalent incidence of the major Ig classes. Deposits in patients with blisters were slightly more intense and a linear pattern of fluorescence seen in 75% of these patients was not seen in controls. The leukocyte attachment assay showed significantly greater (p less than .02) cell attachment in patients with blisters (mean = 167) than in patients without blisters (mean = 64) and greater cell attachment in peribullous than normal skin from the same patient. Organ culture showed complement-dependent migration of leukocytes and histologic features similar to those in spontaneous lesions in skin from the patient with blisters but not in skin from the patient without blisters. These results provide evidence supporting immune complex and complement-dependent inflammation in the pathogenesis of bullous lesions in systemic lupus erythematosus.

Adult↗
PubMed1992

The role of the load and duration of Schistosoma mansoni infection in immune complex mediated schistosomal nephropathy in hamsters.

A significant proteinuria, impairment renal function tests and increased Schistosomal immune complex were observed in infected hamsters by injection of 150 cercariae or more but not in the controls and the hamsters infected with 100 cercariae. There was a significant correlation between these changes and duration of infection and the number of adult worm recovered from the mesenteric circulation Histopathological evaluation showed appearance of circulating immune complex and IgG deposits by the 9th-week following infection, mesangial proliferative glomeriolonephritis appeared in the renal biopsies. There was a significant correlation between the pathologic changes and duration and the load of S. mansoni infection. It is clear that both immune complex deposition and amyloidosis stand as major pathogenic mechanisms that deposited in mesangium and along peripheral capillary lining.

Animals↗
PubMed2000

C5b-9 membrane attack complex mediates endothelial cell apoptosis in experimental glomerulonephritis.

We studied the role of the C5b-9 membrane attack complex in two models of inflammatory glomerulonephritis (GN) initiated by acute glomerular endothelial injury in Piebold-viral-Glaxo (PVG) complement-sufficient rats (C+), C6-deficient rats (C6-), and rats systematically depleted of complement with cobra venom factor (CVF). GN was induced by performing a left nephrectomy and selectively perfusing the right kidney with either 1) the lectin concanavalin A (Con A) followed by complement-fixing anti-Con A (Con A GN) or 2) purified complement-fixing goat anti-rat glomerular endothelial cell (GEN) antibody [immune-mediated thrombotic microangiopathy (ITM)]. Comparable levels of GEN apoptosis were detected in C+ animals in both models. CVF administration reduced GEN apoptosis by 10- to 12-fold. GEN apoptosis was C5b-9 dependent because PVG C6- rats were protected from GEN loss. Furthermore, functional inhibition of the cell surface complement regulatory protein CD59 by renal perfusion with anti-CD59 antibody in ITM resulted in a 3.5-fold increase in GEN apoptosis. Last, in Con A GN, abrogation of GEN apoptosis preserved endothelial integrity and renal function. This study demonstrates the specific role of C5b-9 in the induction of GEN apoptosis in experimental inflammatory GN, a finding with implications for diseases associated with the presence of antiendothelial cell antibodies.

Animals↗
PubMed2006

Quantitative proteomic analysis of distinct mammalian Mediator complexes using normalized spectral abundance factors.

Components of multiprotein complexes are routinely determined by using proteomic approaches. However, this information lacks functional content except when new complex members are identified. To analyze quantitatively the abundance of proteins in human Mediator we used normalized spectral abundance factors generated from shotgun proteomics data sets. With this approach we define a common core of mammalian Mediator subunits shared by alternative forms that variably associate with the kinase module and RNA polymerase (pol) II. Although each version of affinity-purified Mediator contained some kinase module and RNA pol II, Mediator purified through F-Med26 contained the most RNA pol II and the least kinase module as demonstrated by the normalized spectral abundance factor approach. The distinct forms of Mediator were functionally characterized by using a transcriptional activity assay, where F-Med26 Mediator/RNA pol II was the most active. This method of protein complex visualization has important implications for the analysis of multiprotein complexes and assembly of protein interaction networks.

HeLa Cells↗
PubMed1998

The Arp2/3 complex mediates actin polymerization induced by the small GTP-binding protein Cdc42.

The small GTP-binding protein Cdc42 is thought to induce filopodium formation by regulating actin polymerization at the cell cortex. Although several Cdc42-binding proteins have been identified and some of them have been implicated in filopodium formation, the precise role of Cdc42 in modulating actin polymerization has not been defined. To understand the biochemical pathways that link Cdc42 to the actin cytoskeleton, we have reconstituted Cdc42-induced actin polymerization in Xenopus egg extracts. Using this cell-free system, we have developed a rapid and specific assay that has allowed us to fractionate the extract and isolate factors involved in this activity. We report here that at least two biochemically distinct components are required, based on their chromatographic behavior and affinity for Cdc42. One component is purified to homogeneity and is identified as the Arp2/3 complex, a protein complex that has been shown to nucleate actin polymerization. However, the purified complex alone is not sufficient to mediate the activity; a second component that binds Cdc42 directly and mediates the interaction between Cdc42 and the complex also is required. These results establish an important link between a signaling molecule, Cdc42, and a complex that can directly modulate actin networks in vitro. We propose that activation of the Arp2/3 complex by Cdc42 and other signaling molecules plays a central role in stimulating actin polymerization at the cell surface.

Actin-Related Protein 2↗
PubMed2004

Members of the SAGA and Mediator complexes are partners of the transcription elongation factor TFIIS.

TFIIS, an elongation factor encoded by DST1 in Saccharomyces cerevisiae, stimulates transcript cleavage in arrested RNA polymerase II. Two components of the RNA polymerase II machinery, Med13 (Srb9) and Spt8, were isolated as two-hybrid partners of the conserved TFIIS N-terminal domain. They belong to the Cdk8 module of the Mediator and to a subform of the SAGA co-activator, respectively. Co-immunoprecipitation experiments showed that TFIIS can bind the Cdk8 module and SAGA in cell-free extracts. spt8Delta and dst1Delta mutants were sensitive to nucleotide-depleting drugs and epistatic to null mutants of the RNA polymerase II subunit Rpb9, suggesting that their elongation defects are mediated by Rpb9. rpb9Delta, spt8Delta and dst1Delta were lethal in cells lacking the Rpb4 subunit. The TFIIS N-terminal domain is also strictly required for viability in rpb4Delta, although it is not needed for binding to RNA polymerase II or for transcript cleavage. It is proposed that TFIIS and the Spt8-containing form of SAGA co-operate to rescue RNA polymerase II from unproductive elongation complexes, and that the Cdk8 module temporarily blocks transcription during transcript cleavage.

Amino Acid Sequence↗
PubMed2006

COG complex-mediated recycling of Golgi glycosyltransferases is essential for normal protein glycosylation.

Defects in conserved oligomeric Golgi (COG) complex result in multiple deficiencies in protein glycosylation. On the other hand, acute knock-down (KD) of Cog3p (COG3 KD) causes accumulation of intra-Golgi COG complex-dependent (CCD) vesicles. Here, we analyzed cellular phenotypes at different stages of COG3 KD to uncover the molecular link between COG function and glycosylation disorders. For the first time, we demonstrated that medial-Golgi enzymes are transiently relocated into CCD vesicles in COG3 KD cells. As a result, Golgi modifications of both plasma membrane (CD44) and lysosomal (Lamp2) glycoproteins are distorted. Localization of these proteins is not altered, indicating that the COG complex is not required for anterograde trafficking and accurate sorting. COG7 KD and double COG3/COG7 KD caused similar defects with respect to both Golgi traffic and glycosylation, suggesting that the entire COG complex orchestrates recycling of medial-Golgi-resident proteins. COG complex-dependent docking of isolated CCD vesicles was reconstituted in vitro, supporting their role as functional trafficking intermediates. Altogether, the data suggest that constantly cycling medial-Golgi enzymes are transported from distal compartments in CCD vesicles. Dysfunction of COG complex leads to separation of glycosyltransferases from anterograde cargo molecules passing along secretory pathway, thus affecting normal protein glycosylation.

Adaptor Proteins, Vesicular Transport↗
PubMed2006

Supramolecular complexes mediate selenocysteine incorporation in vivo.

Selenocysteine incorporation in eukaryotes occurs cotranslationally at UGA codons via the interactions of RNA-protein complexes, one comprised of selenocysteyl (Sec)-tRNA([Ser]Sec) and its specific elongation factor, EFsec, and another consisting of the SECIS element and SECIS binding protein, SBP2. Other factors implicated in this pathway include two selenophosphate synthetases, SPS1 and SPS2, ribosomal protein L30, and two factors identified as binding tRNA([Ser]Sec), termed soluble liver antigen/liver protein (SLA/LP) and SECp43. We report that SLA/LP and SPS1 interact in vitro and in vivo and that SECp43 cotransfection increases this interaction and redistributes all three proteins to a predominantly nuclear localization. We further show that SECp43 interacts with the selenocysteyl-tRNA([Ser]Sec)-EFsec complex in vitro, and SECp43 coexpression promotes interaction between EFsec and SBP2 in vivo. Additionally, SECp43 increases selenocysteine incorporation and selenoprotein mRNA levels, the latter presumably due to circumvention of nonsense-mediated decay. Thus, SECp43 emerges as a key player in orchestrating the interactions and localization of the other factors involved in selenoprotein biosynthesis. Finally, our studies delineating the multiple, coordinated protein-nucleic acid interactions between SECp43 and the previously described selenoprotein cotranslational factors resulted in a model of selenocysteine biosynthesis and incorporation dependent upon both cytoplasmic and nuclear supramolecular complexes.

Autoantigens↗
PubMed2003

A dynamin-cortactin-Arp2/3 complex mediates actin reorganization in growth factor-stimulated cells.

The mechanisms by which mammalian cells remodel the actin cytoskeleton in response to motogenic stimuli are complex and a topic of intense study. Dynamin 2 (Dyn2) is a large GTPase that interacts directly with several actin binding proteins, including cortactin. In this study, we demonstrate that Dyn2 and cortactin function to mediate dynamic remodeling of the actin cytoskeleton in response to stimulation with the motogenic growth factor platelet-derived growth factor. On stimulation, Dyn2 and cortactin coassemble into large, circular structures on the dorsal cell surface. These "waves" promote an active reorganization of actin filaments in the anterior cytoplasm and function to disassemble actin stress fibers. Importantly, inhibition of Dyn2 and cortactin function potently blocked the formation of waves and subsequent actin reorganization. These findings demonstrate that cortactin and Dyn2 function together in a supramolecular complex that assembles in response to growth factor stimulation and mediates the remodeling of actin to facilitate lamellipodial protrusion at the leading edge of migrating cells.

Actin-Related Protein 3↗
PubMed1999

Trophinin, tastin, and bystin: a complex mediating unique attachment between trophoblastic and endometrial epithelial cells at their respective apical cell membranes.

Embryo implantation is a complex process consisting of multiple cross-talks between maternal and embryonic cells. Defining the mechanisms underlying implantation at molecular level is challenging task in reproductive biology. In order to identify molecules involved in cellular interactions between trophoblastic and endometrial epithelial cells, we have established two human cell lines, trophoblastic HT-H and endometrial epithelial SNGM. These two cell types exhibit cell adhesion at their respective apical cell membranes. Molecules involved in this unique cell adhesion were identified by expression complementary DNA cloning and were named trophinin, tastin, and bystin. Trophinin is a membrane protein thought to have self-binding activity and thus mediates homophilic cell adhesion. Tastin and bystin are cytoplasmic proteins required for trophinin to exhibit cell adhesion activity. Trophinin is strongly expressed in trophectoderm of monkey blastocysts. In human endometrium, trophinin is expressed for a limited period in the luminal epithelium at the time expected for implantation. In human placenta, trophinin, tastin, and bystin are strongly expressed in trophoblast and endometrium at the uteroplacental interface at an early stage in pregnancy. All these molecules disappear from the human placenta in the second trimester. The unique expression pattern and cell adhesion activity exhibited by trophinin, tastin, and bystin suggest strongly the involvement of these molecules in the initial attachment of blastocyst to uterus.

Animals↗
PubMed1981

Phagocytosis of immune complexes mediated by IgM and C3 receptors by macrophages from mice treated with glycogen.

Phagocytosis mediated by IgM and C3 receptors was studied by in vitro experiments with peritoneal macrophages from mice treated with repeated i.p. injections of glycogen. The immune complexes were sheep red cells sensitized with mouse IgM antibody or antibody and complement. Macrophages from the inflammatory exudate induced by glycogen were able to phagocytize immune complexes of red cells and IgM antibodies in contrast to normal macrophages, which can only bind to these complexes. Evidence was also found for phagocytosis mediated by interaction with C3 receptors in the macrophages from the glycogen-treated animals. The implications of these findings in the mechanism of phagocytosis and the possible relevance of the observed changes in the function of macrophage receptors for the afferent and efferent branches of immune response are discussed.

Animals↗