PubMed Health⌕ Search

Biomedical subjects

Paul Tempst

Publications and source records attributed to Paul Tempst.

At least 73 records · Page 4Linked to original sources
PubMed2004

The Yaf9 component of the SWR1 and NuA4 complexes is required for proper gene expression, histone H4 acetylation, and Htz1 replacement near telomeres.

Yaf9, Taf14, and Sas5 comprise the YEATS domain family in Saccharomyces cerevisiae, which in humans includes proteins involved in acute leukemias. The YEATS domain family is essential, as a yaf9Delta taf14Delta sas5Delta triple mutant is nonviable. We verify that Yaf9 is a stable component of NuA4, an essential histone H4 acetyltransferase complex. Yaf9 is also associated with the SWR1 complex, which deposits the histone H2A variant Htz1. However, the functional contribution of Yaf9 to these complexes has not been determined. Strains lacking YAF9 are sensitive to DNA-damaging agents, cold, and caffeine, and the YEATS domain is required for full Yaf9 function. NuA4 lacking Yaf9 retains histone acetyltransferase activity in vitro, and Yaf9 does not markedly reduce bulk H4 acetylation levels, suggesting a role for Yaf9 in the targeting or regulation of NuA4. Interestingly, yaf9Delta strains display reduced transcription of genes near certain telomeres, and their repression is correlated with reduced H4 acetylation, reduced occupancy by Htz1, and increased occupancy by the silencing protein Sir3. Additionally, the spectra of phenotypes, genes, and telomeres affected in yaf9Delta and htz1Delta strains are significantly similar, further supporting a role for Yaf9 in Htz1 deposition. Taken together, these data indicate that Yaf9 may function in NuA4 and SWR1 complexes to help antagonize silencing near telomeres.

Acetylation↗
PubMed2004

Human SWI/SNF-associated PRMT5 methylates histone H3 arginine 8 and negatively regulates expression of ST7 and NM23 tumor suppressor genes.

Protein arginine methyltransferases (PRMTs) have been implicated in transcriptional activation and repression, but their role in controlling cell growth and proliferation remains obscure. We have recently shown that PRMT5 can interact with flag-tagged BRG1- and hBRM-based hSWI/SNF chromatin remodelers and that both complexes can specifically methylate histones H3 and H4. Here we report that PRMT5 can be found in association with endogenous hSWI/SNF complexes, which can methylate H3 and H4 N-terminal tails, and show that H3 arginine 8 and H4 arginine 3 are preferred sites of methylation by recombinant and hSWI/SNF-associated PRMT5. To elucidate the role played by PRMT5 in gene regulation, we have established a PRMT5 antisense cell line and determined by microarray analysis that more genes are derepressed when PRMT5 levels are reduced. Among the affected genes, we show that suppressor of tumorigenicity 7 (ST7) and nonmetastatic 23 (NM23) are direct targets of PRMT5-containing BRG1 and hBRM complexes. Furthermore, we demonstrate that expression of ST7 and NM23 is reduced in a cell line that overexpresses PRMT5 and that this decrease in expression correlates with H3R8 methylation, H3K9 deacetylation, and increased transformation of NIH 3T3 cells. These findings suggest that the BRG1- and hBRM-associated PRMT5 regulates cell growth and proliferation by controlling expression of genes involved in tumor suppression.

Acetylation↗
PubMed2003

Affinity capture of specific DNA-binding proteins for mass spectrometric identification.

We describe a general approach for affinity microcapture of site-specific, nucleic acid-binding proteins. The major difficulties to developing this method into a widely applicable protocol derived from the need for a massive enrichment and the inadvertent, extensive binding of nonspecific proteins to the bait. On the basis of a detailed analysis, we propose (i) a one-step fractionation of crude extracts on P11 phosphocellulose, followed by (ii) a discrete series of positive/negative selections on wild-type and site-mutated ligand DNA in a magnetic microparticulate format, with cobalt magnets, concatamerized and biotinylated ligands, selective salt conditions, and improved competitor DNAs. We also present rules for determining the precise number and order of selections. The approach and protocol allowed isolation of four, low-abundance transcription factors and repressors from 2 x 10(9) cultured leukemia cells. Captured proteins were 10-20,000-fold enriched from the nuclear extract, in a form and amounts that permitted facile MALDI-TOF and TOF/TOF MS-based protein identification. This is 1-2 orders of magnitude better than many previous efforts and in a fraction of the time (approximately 1 factor/week). The method can be applied to any protein that binds DNA, including those with modest to low affinity, and bridges functional-biochemical studies on replication, transcriptional regulation, and DNA repair with the analytical power of mass spectrometry-based proteomics.

Cell Line, Tumor↗
PubMed2003

The laminin receptor modulates granulocyte-macrophage colony-stimulating factor receptor complex formation and modulates its signaling.

Basement membrane matrix proteins are known to up-regulate granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling in neutrophils and mononuclear phagocytes, but the mechanisms involved are poorly understood. We used the intracellular portion of the alpha subunit of the GM-CSF receptor (alphaGMR) to search for interacting proteins and identified the 67-kDa laminin receptor (LR), a nonintegrin matrix protein receptor expressed in several types of host defense cells and certain tumors, as a binding partner. LR was found to interact with the beta subunit of the GMR (betaGMR) as well. Whereas GM-CSF functions by engaging the alphaGMR and betaGMR into receptor complexes, LR inhibited GM-CSF-induced receptor complex formation. Laminin and fibronectin binding to LR was found to prevent the binding of betaGMR to LR and relieved the LR inhibition of GMR. These findings provide a mechanistic basis for enhancing host defense cell responsiveness to GM-CSF at transendothelial migration sites while suppressing it in circulation.

Base Sequence↗
PubMed2003

Revised subunit structure of yeast transcription factor IIH (TFIIH) and reconciliation with human TFIIH.

Tfb4 is identified as a subunit of the core complex of yeast RNA polymerase II general transcription factor IIH (TFIIH) by affinity purification, by peptide sequence analysis, and by expression of the entire complex in insect cells. Tfb3, previously identified as a component of the core complex, is shown instead to form a complex with cdk and cyclin subunits of TFIIH. This reassignment of subunits resolves a longstanding discrepancy between yeast and human TFIIH complexes.

Amino Acid Sequence↗
PubMed2003

The budding yeast Rad9 checkpoint complex: chaperone proteins are required for its function.

Rad9 functions in the DNA-damage checkpoint pathway of Saccharomyces cerevisiae. In whole-cell extracts, Rad9 is found in large, soluble complexes, which have functions in amplifying the checkpoint signal. The two main soluble forms of Rad9 complexes that are found in cells exposed to DNA-damaging treatments were purified to homogeneity. Both of these Rad9 complexes contain the Ssa1 and/or Ssa2 chaperone proteins, suggesting a function for these proteins in checkpoint regulation. Consistent with this possibility, genetic experiments indicate redundant functions for SSA1 and SSA2 in survival, G2/M-checkpoint regulation, and phosphorylation of both Rad9 and Rad53 after irradiation with ultraviolet light. Ssa1 and Ssa2 can now be considered as novel checkpoint proteins that are likely to be required for remodelling Rad9 complexes during checkpoint-pathway activation.

Adenosine Triphosphatases↗
PubMed2003

Nab2p and the Thp1p-Sac3p complex functionally interact at the interface between transcription and mRNA metabolism.

THP1 is a conserved eukaryotic gene whose null mutations confer, in yeast, transcription and genetic instability phenotypes and RNA export defects similar to those of the THO/TREX complex null mutations. In a search for multicopy suppressors of the transcription defect of thp1Delta cells, we identified the poly(A)+ RNA-binding heterogeneous nuclear ribonucleoprotein Nab2p. Multicopy NAB2 also suppressed the RNA export defect of thp1Delta cells. This result suggests a functional relationship between Thp1p and Nab2p. Consistently, the leaky mutation nab2-1 conferred a transcription defect and hyper-recombination phenotype similar to those of thp1Delta, although to a minor degree. Reciprocally, a purified His6-tagged Thp1p fusion bound RNA in vitro. In a different approach, we show by Western analyses that a highly purified Thp1p-Sac3p complex does not contain components of THO/TREX and that sac3Delta confers a transcription defect and hyper-recombination phenotype identical to those of thp1Delta. mRNA degradation was not affected in thp1Delta mutants, implying that their expression defects are not due to mRNA decay. This indicates that Thp1p-Sac3p is a structural and functional unit. Altogether, our results suggest that Thp1p-Sac3p and Nab2p are functionally related heterogeneous nuclear ribonucleoproteins that define a further link between mRNA metabolism and transcription.

Fungal Proteins↗
PubMed2003

Catalytic properties of ADAM19.

ADAMs are membrane-anchored glycoproteins with functions in fertilization, heart development, neurogenesis, and protein ectodomain shedding. Here we report an evaluation of the catalytic activity of recombinantly expressed soluble forms of ADAM19, a protein that is essential for cardiovascular morphogenesis. Proteolytic activity of soluble forms of ADAM19 was first demonstrated by their autocatalytic removal of a purification tag (Myc-His) and their ability to cleave myelin basic protein and the insulin B chain. The metalloprotease activity of ADAM19 is sensitive to the hydroxamic acid-type metalloprotease inhibitor BB94 (batimastat) but not to tissue inhibitors of metalloproteases (TIMPs) 1-3. Moreover, ADAM19 cleaves peptides corresponding to the known cleavage sites of tumor necrosis factor-alpha (TNF-alpha), TNF-related activation-induced cytokine (TRANCE, also referred to as osteoprotegerin ligand), and kit ligand-1 (KL-1) in vitro. Although ADAM19 is not required for shedding of TNFalpha and TRANCE in mouse embryonic fibroblasts, its overexpression in COS-7 cells results in strongly increased TRANCE shedding. This suggests a potential role for ADAM19 in shedding TRANCE in cells where both molecules are highly expressed, such as in osteoblasts. Interestingly, our results also indicate that ADAM19 can function as a negative regulator of KL-1 shedding in both COS-7 cells and mouse embryonic fibroblasts, instead of acting directly on KL-1. The identification of potential in vitro substrates offers the basis for further functional studies of ADAM19 in cells and in mice.

ADAM Proteins↗
PubMed2003

Parkinson's disease-associated alpha-synuclein is a calmodulin substrate.

Alpha-synuclein is a neuronal protein thought to be central in the pathogenesis of Parkinson's disease (PD) because it comprises the fibrillar core of Lewy bodies, one of the histologically defining lesions of PD, and because mutations in alpha-synuclein cause autosomal dominant PD. Although its physiologic role is uncertain, alpha-synuclein is a synaptic protein that may contribute to plasticity. We produced synuclein with incorporated photoprobes to identify and purify novel synuclein-interacting proteins both to begin to clarify the physiology of synuclein and to identify factors that may regulate synuclein conformation. We detected several cross-links and purified and identified one as calmodulin (CaM). CaM binds to both wild type and PD-associated mutant alpha-synucleins in a calcium-dependent manner. We further demonstrate that CaM and alpha-synuclein interact in intact cells in a calcium-dependent manner and that activated CaM accelerates the formation of synuclein fibrils in vitro. We hypothesize that the known calcium control of synuclein function is mediated through CaM interaction and that CaM potentially alters synuclein conformation.

Calmodulin↗
PubMed2003

The C-terminal domain phosphatase and transcription elongation activities of FCP1 are regulated by phosphorylation.

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) is heavily phosphorylated during the transition from transcription initiation to the establishment of an elongation-competent transcription complex. FCP1 is the only phosphatase known to be specific for the CTD of the largest subunit of RNAPII, and its activity is believed to be required to reactivate RNAPII, so that RNAPII can enter another round of transcription. We demonstrate that FCP1 is a phosphoprotein, and that phosphorylation regulates FCP1 activities. FCP1 is phosphorylated at multiple sites in vivo. The CTD phosphatase activity of phosphorylated FCP1 is stimulated by TFIIF, whereas dephosphorylated FCP1 is not. In addition to its role in the recycling of RNAPII, FCP1 also affects transcription elongation. Phosphorylated FCP1 is more active in stimulating transcription elongation than the dephosphorylated form of FCP1. We found that only phosphorylated FCP1 can physically interact with TFIIF. We set out to purify an FCP1 kinase from HeLa cells and identified casein kinase 2, which, surprisingly, displayed a negative effect on FCP1-associated activities.

Alkaline Phosphatase↗
PubMed2003

mAM facilitates conversion by ESET of dimethyl to trimethyl lysine 9 of histone H3 to cause transcriptional repression.

Methylation of histone tails plays an important role in chromatin structure and function. Previously, we reported that ESET/SETDB1 is a histone methyltransferase (HMTase). Here, we show that SETDB1 tightly associates with the human homolog of mAM, a murine ATFa-associated factor. Although recombinant ESET can methylate lysine 9 of histone H3 (H3-K9), its activity is severely compromised when compared to that of the ESET/mAM complex. mAM stimulates ESET enzymatic activity by increasing the Vmax and decreasing the Km. Importantly, mAM facilitates the ESET-dependent conversion of dimethyl H3-K9 to the trimethyl state both in vitro and in vivo. Chromatin-based transcription and ChIP analyses demonstrate that mAM enhances ESET-mediated transcriptional repression in a SAM-dependent manner, and this repression correlates with H3-K9 trimethylation at the promoter. Thus, our studies establish that promoter H3-K9 trimethylation is the cause of transcriptional repression and that mAM/hAM facilitates conversion of H3-K9 dimethyl to trimethyl by ESET/SETDB1.

Amino Acid Sequence↗
PubMed2003

GbetaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR.

mTOR and raptor are components of a signaling pathway that regulates mammalian cell growth in response to nutrients and growth factors. Here, we identify a member of this pathway, a protein named GbetaL that binds to the kinase domain of mTOR and stabilizes the interaction of raptor with mTOR. Like mTOR and raptor, GbetaL participates in nutrient- and growth factor-mediated signaling to S6K1, a downstream effector of mTOR, and in the control of cell size. The binding of GbetaL to mTOR strongly stimulates the kinase activity of mTOR toward S6K1 and 4E-BP1, an effect reversed by the stable interaction of raptor with mTOR. Interestingly, nutrients and rapamycin regulate the association between mTOR and raptor only in complexes that also contain GbetaL. Thus, we propose that the opposing effects on mTOR activity of the GbetaL- and raptor-mediated interactions regulate the mTOR pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Proteolytic cleavage of MLL generates a complex of N- and C-terminal fragments that confers protein stability and subnuclear localization.

The mixed-lineage leukemia gene (MLL, ALL1, HRX) encodes a 3,969-amino-acid nuclear protein homologous to Drosophila trithorax and is required to maintain proper Hox gene expression. Chromosome translocations in human leukemia disrupt MLL (11q23), generating chimeric proteins between the N terminus of MLL and multiple translocation partners. Here we report that MLL is normally cleaved at two conserved sites (D/GADD and D/GVDD) and that mutation of these sites abolishes the proteolysis. MLL cleavage generates N-terminal p320 (N320) and C-terminal p180 (C180) fragments, which form a stable complex that localizes to a subnuclear compartment. The FYRN domain of N320 directly interacts with the FYRC and SET domains of C180. Disrupting the interaction between N320 and C180 leads to a marked decrease in the level of N320 and a redistribution of C180 to a diffuse nuclear pattern. These data suggest a model in which a dynamic post-cleavage association confers stability to N320 and correct nuclear sublocalization of the complex, to control the availability of N320 for target genes. This predicts that MLL fusion proteins of leukemia which would lose the ability to complex with C180 have their stability conferred instead by the fusion partners, thus providing one mechanism for altered target gene expression.

Amino Acid Motifs↗
PubMed2003

mSin3A/histone deacetylase 2- and PRMT5-containing Brg1 complex is involved in transcriptional repression of the Myc target gene cad.

The role of hSWI/SNF complexes in transcriptional activation is well characterized; however, little is known about their function in transcriptional repression. We have previously shown that subunits of the mSin3A/histone deacetylase 2 (HDAC2) corepressor complex copurify with hSWI/SNF complexes. Here we show that the type II arginine-specific methyltransferase PRMT5, which is involved in cyclin E repression, can be found in association with Brg1 and hBrm-based hSWI/SNF complexes. We also show that hSWI/SNF-associated PRMT5 can methylate hypoacetylated histones H3 and H4 more efficiently than hyperacetylated histones H3 and H4. Protein-protein interaction studies indicate that PRMT5 and mSin3A interact with the same hSWI/SNF subunits as those targeted by c-Myc. These observations prompted us to examine the expression profile of the c-Myc target genes, carbamoyl-phosphate synthase-aspartate carbamoyltransferase-dihydroorotase (cad) and nucleolin (nuc). We found that cad repression is altered in cells that express inactive Brg1 and in cells treated with the HDAC inhibitor depsipeptide. Using chromatin immunoprecipitation assays, we found that Brg1, mSin3A, HDAC2, and PRMT5 are directly recruited to the cad promoter. These results suggest that hSWI/SNF complexes, through their ability to interact with activator and repressor proteins, control expression of genes involved in cell growth and proliferation.

Aspartate Carbamoyltransferase↗
PubMed2003

ASAP, a novel protein complex involved in RNA processing and apoptosis.

Different isoforms of a protein complex termed the apoptosis- and splicing-associated protein (ASAP) were isolated from HeLa cell extract. ASAP complexes are composed of the polypeptides SAP18 and RNPS1 and different isoforms of the Acinus protein. While Acinus had previously been implicated in apoptosis and was recently identified as a component of the spliceosome, RNPS1 has been described as a general activator of RNA processing. Addition of ASAP isoforms to in vitro splicing reactions inhibits RNA processing mediated by ASF/SF2, by SC35, or by RNPS1. Additionally, microinjection of ASAP complexes into mammalian cells resulted in acceleration of cell death. Importantly, after induction of apoptosis the ASAP complex disassembles. Taken together, our results suggest an important role for the ASAP complexes in linking RNA processing and apoptosis.

Apoptosis↗
PubMed2002

Conversion of proepithelin to epithelins: roles of SLPI and elastase in host defense and wound repair.

Increased leukocyte elastase activity in mice lacking secretory leukocyte protease inhibitor (SLPI) leads to impaired wound healing due to enhanced activity of TGFbeta and perhaps additional mechanisms. Proepithelin (PEPI), an epithelial growth factor, can be converted to epithelins (EPIs) in vivo by unknown mechanisms with unknown consequences. We found that PEPI and EPIs exert opposing activities. EPIs inhibit the growth of epithelial cells but induce them to secrete the neutrophil attractant IL-8, while PEPI blocks neutrophil activation by tumor necrosis factor, preventing release of oxidants and proteases. SLPI and PEPI form complexes, preventing elastase from converting PEPI to EPIs. Supplying PEPI corrects the wound-healing defect in SLPI null mice. Thus, SLPI/elastase act via PEPI/EPIs to operate a switch at the interface between innate immunity and wound healing.

Amino Acid Sequence↗
PubMed2002

Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein.

Enhancer of Zeste [E(z)] is a Polycomb-group transcriptional repressor and one of the founding members of the family of SET domain-containing proteins. Several SET-domain proteins possess intrinsic histone methyltransferase (HMT) activity. However, recombinant E(z) protein was found to be inactive in a HMT assay. Here we report the isolation of a multiprotein E(z) complex that contains extra sex combs, suppressor of zeste-12 [Su(z)12], and the histone binding proteins RbAp46/RbAp48. This complex, which we termed Polycomb repressive complex (PRC) 2, possesses HMT activity with specificity for Lys 9 (K9) and Lys 27 (K27) of histone H3. The HMT activity of PRC2 is dependent on an intact SET domain in the E(z) protein. We hypothesize that transcriptional repression by the E(z) protein involves methylation-dependent recruitment of PRC1. The presence of Su(z)12, a strong suppressor of position effect variegation, in PRC2 suggests that PRC2 may play a widespread role in heterochromatin-mediated silencing.

Amino Acid Sequence↗