RESUMO
The conserved transcriptional regulator heat shock factor 1 (Hsf1) is a key sensor of proteotoxic and other stress in the eukaryotic cytosol. We surveyed Hsf1 activity in a genome-wide loss-of-function library in Saccaromyces cerevisiae as well as ~78,000 double mutants and found Hsf1 activity to be modulated by highly diverse stresses. These included disruption of a ribosome-bound complex we named the Ribosome Quality Control Complex (RQC) comprising the Ltn1 E3 ubiquitin ligase, two highly conserved but poorly characterized proteins (Tae2 and Rqc1), and Cdc48 and its cofactors. Electron microscopy and biochemical analyses revealed that the RQC forms a stable complex with 60S ribosomal subunits containing stalled polypeptides and triggers their degradation. A negative feedback loop regulates the RQC, and Hsf1 senses an RQC-mediated translation-stress signal distinctly from other stresses. Our work reveals the range of stresses Hsf1 monitors and elucidates a conserved cotranslational protein quality control mechanism.
Assuntos
Complexos Multiproteicos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Choque Térmico/genética , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Ligação a RNA , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteína com ValosinaRESUMO
Toxins have evolved to target regions of membrane ion channels that underlie ligand binding, gating, or ion permeation, and have thus served as invaluable tools for probing channel structure and function. Here, we describe a peptide toxin from the Earth Tiger tarantula that selectively and irreversibly activates the capsaicin- and heat-sensitive channel, TRPV1. This high-avidity interaction derives from a unique tandem repeat structure of the toxin that endows it with an antibody-like bivalency. The "double-knot" toxin traps TRPV1 in the open state by interacting with residues in the presumptive pore-forming region of the channel, highlighting the importance of conformational changes in the outer pore region of TRP channels during activation.
Assuntos
Venenos de Aranha/metabolismo , Canais de Cátion TRPV/metabolismo , Proteínas de Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Fenômenos Eletrofisiológicos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Neurônios/metabolismo , Oócitos/metabolismo , Ratos , Venenos de Aranha/química , Canais de Cátion TRPV/química , Gânglio Trigeminal/citologia , Gânglio Trigeminal/metabolismo , Proteínas de Xenopus/químicaRESUMO
Natural products that elicit discomfort or pain represent invaluable tools for probing molecular mechanisms underlying pain sensation. Plant-derived irritants have predominated in this regard, but animal venoms have also evolved to avert predators by targeting neurons and receptors whose activation produces noxious sensations. As such, venoms provide a rich and varied source of small molecule and protein pharmacophores that can be exploited to characterize and manipulate key components of the pain-signalling pathway. With this in mind, here we perform an unbiased in vitro screen to identify snake venoms capable of activating somatosensory neurons. Venom from the Texas coral snake (Micrurus tener tener), whose bite produces intense and unremitting pain, excites a large cohort of sensory neurons. The purified active species (MitTx) consists of a heteromeric complex between Kunitz- and phospholipase-A2-like proteins that together function as a potent, persistent and selective agonist for acid-sensing ion channels (ASICs), showing equal or greater efficacy compared with acidic pH. MitTx is highly selective for the ASIC1 subtype at neutral pH; under more acidic conditions (pH < 6.5), MitTx massively potentiates (>100-fold) proton-evoked activation of ASIC2a channels. These observations raise the possibility that ASIC channels function as coincidence detectors for extracellular protons and other, as yet unidentified, endogenous factors. Purified MitTx elicits robust pain-related behaviour in mice by activation of ASIC1 channels on capsaicin-sensitive nerve fibres. These findings reveal a mechanism whereby snake venoms produce pain, and highlight an unexpected contribution of ASIC1 channels to nociception.
Assuntos
Venenos Elapídicos/química , Venenos Elapídicos/farmacologia , Elapidae , Proteínas do Tecido Nervoso/metabolismo , Dor/induzido quimicamente , Multimerização Proteica , Canais de Sódio/metabolismo , Canais Iônicos Sensíveis a Ácido , Sequência de Aminoácidos , Animais , Capsaicina/farmacologia , Células Cultivadas , Membro Posterior/efeitos dos fármacos , Membro Posterior/fisiopatologia , Humanos , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Nociceptividade/efeitos dos fármacos , Nociceptividade/fisiologia , Oócitos , Dor/metabolismo , Dor/fisiopatologia , Estrutura Quaternária de Proteína , Prótons , Ratos , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Agonistas de Canais de Sódio , Canais de Sódio/deficiência , Canais de Sódio/genética , Canais de Cátion TRPV/metabolismo , Xenopus laevisRESUMO
Uridine insertion/deletion RNA editing in kinetoplastid mitochondria corrects encoded frameshifts in mRNAs. The genetic information for editing resides in small guide RNAs (gRNAs), which form anchor duplexes just downstream of an editing site and mediate editing within a single editing "block." Many mRNAs require multiple gRNAs; the observed overall 3' to 5' polarity of editing is determined by the formation of upstream mRNA anchors by downstream editing. Hel61, a mitochondrial DEAD-box protein, was previously shown to be involved in RNA editing, but the functional role was not clear. Here we report that down-regulation of Hel61 [renamed REH1 (RNA editing helicase 1)] expression in Trypanosoma brucei selectively affects editing mediated by two or more overlapping gRNAs but has no effect on editing within a single block. Down-regulation produces an increased abundance of the gRNA/edited mRNA duplex for the first editing block of the A6 mRNA. Recombinant REH1 has an ATP-dependent double strand RNA unwinding activity in vitro with a model gRNA-mRNA duplex. These data indicate that REH1 is involved in gRNA displacement either directly by unwinding the gRNA/edited mRNA duplex or indirectly, to allow the 5' adjacent upstream gRNA to form an anchor duplex with the edited mRNA to initiate another block of editing. Purified tagged REH1 is associated with the RNA editing core complex by RNA linkers and a colocalization of REH1, REL1, and two kinetoplast ribosomal proteins with the kinetoplast DNA was observed by immunofluorescence, suggesting that editing, transcription, and translation may be functionally linked.
Assuntos
Mutação INDEL/genética , Proteínas de Protozoários/metabolismo , Edição de RNA/genética , RNA Helicases/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Trypanosoma brucei brucei/enzimologia , Uridina/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Regulação para Baixo/genética , Espaço Intracelular/metabolismo , Mitocôndrias/metabolismo , Transporte Proteico , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/metabolismo , Trypanosoma brucei brucei/genéticaRESUMO
Bites and stings from venomous creatures can produce pain and inflammation as part of their defensive strategy to ward off predators or competitors. Molecules accounting for lethal effects of venoms have been extensively characterized, but less is known about the mechanisms by which they produce pain. Venoms from spiders, snakes, cone snails or scorpions contain a pharmacopoeia of peptide toxins that block receptor or channel activation as a means of producing shock, paralysis or death. We examined whether these venoms also contain toxins that activate (rather than inhibit) excitatory channels on somatosensory neurons to produce a noxious sensation in mammals. Here we show that venom from a tarantula that is native to the West Indies contains three inhibitor cysteine knot (ICK) peptides that target the capsaicin receptor (TRPV1), an excitatory channel expressed by sensory neurons of the pain pathway. In contrast with the predominant role of ICK toxins as channel inhibitors, these previously unknown 'vanillotoxins' function as TRPV1 agonists, providing new tools for understanding mechanisms of TRP channel gating. Some vanillotoxins also inhibit voltage-gated potassium channels, supporting potential similarities between TRP and voltage-gated channel structures. TRP channels can now be included among the targets of peptide toxins, showing that animals, like plants (for example, chilli peppers), avert predators by activating TRP channels on sensory nerve fibres to elicit pain and inflammation.
Assuntos
Ativação do Canal Iônico/efeitos dos fármacos , Dor/fisiopatologia , Venenos de Aranha/farmacologia , Aranhas/química , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/metabolismo , Animais , Linhagem Celular , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/fisiopatologia , Camundongos , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/metabolismo , Dor/induzido quimicamente , Dor/metabolismo , Técnicas de Patch-Clamp , Ratos , Venenos de Aranha/química , Aranhas/fisiologia , Especificidade por SubstratoRESUMO
Native states of proteins are flexible, populating more than just the unique native conformation. The energetics and dynamics resulting from this conformational ensemble are inherently linked to protein function and regulation. Proteolytic susceptibility is one feature determined by this conformational energy landscape. As an attempt to investigate energetics of proteins on a proteomic scale, we challenged the Escherichia coli proteome with extensive proteolysis and determined which proteins, if any, have optimized their energy landscape for resistance to proteolysis. To our surprise, multiple soluble proteins survived the challenge. Maltose binding protein, a survivor from thermolysin digestion, was characterized by in vitro biophysical studies to identify the physical origin of proteolytic resistance. This experimental characterization shows that kinetic stability is responsible for the unusual resistance in maltose binding protein. The biochemical functions of the identified survivors suggest that many of these proteins may have evolved extreme proteolytic resistance because of their critical roles under stressed conditions. Our results suggest that under functional selection proteins can evolve extreme proteolysis resistance by modulating their conformational energy landscapes without the need to invent new folds, and that proteins can be profiled on a proteomic scale according to their energetic properties by using proteolysis as a structural probe.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Conformação Proteica , Proteoma/análise , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Hidrólise , Proteínas Ligantes de Maltose , Modelos Moleculares , Dados de Sequência Molecular , Termodinâmica , Termolisina/metabolismoRESUMO
The SWI/SNF family of chromatin-remodeling complexes has been discovered in many species and has been shown to regulate gene expression by assisting transcriptional machinery to gain access to their sites in chromatin. Several complexes of this family have been reported for humans. In this study, two additional complexes are described that belong to the same SWI/SNF family. These new complexes contain as many as eight subunits identical to those found in other SWI/SNF complexes, and they possess a similar ATP-dependent nucleosome disruption activity. But unlike known SWI/SNFs, the new complexes are low in abundance and contain an extra subunit conserved between human and yeast SWI/SNF complexes. This subunit, ENL, is a homolog of the yeast SWI/SNF subunit, ANC1/TFG3. Moreover, ENL is a fusion partner for the gene product of MLL that is a common target for chromosomal translocations in human acute leukemia. The resultant MLL-ENL fusion protein associates and cooperates with SWI/SNF complexes to activate transcription of the promoter of HoxA7, a downstream target essential for oncogenic activity of MLL-ENL. Our data suggest that human SWI/SNF complexes show considerable heterogeneity, and one or more may be involved in the etiology of leukemia by cooperating with MLL fusion proteins.
Assuntos
Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Proto-Oncogenes , Fatores de Transcrição/genética , Sequência de Aminoácidos , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Clonagem Molecular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase , Proteínas de Homeodomínio/genética , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide , Proteínas de Neoplasias/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Subunidades Proteicas , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Translocação Genética , Células Tumorais CultivadasRESUMO
CRISPR drives prokaryotic adaptation to invasive nucleic acids such as phages and plasmids, using an RNA-mediated interference mechanism. Interference in type I CRISPR-Cas systems requires a targeting Cascade complex and a degradation machine, Cas3, which contains both nuclease and helicase activities. Here we report the crystal structures of Thermobifida fusca Cas3 bound to single-stranded (ss) DNA substrate and show that it is an obligate 3'-to-5' ssDNase that preferentially accepts substrate directly from the helicase moiety. Conserved residues in the HD-type nuclease coordinate two irons for ssDNA cleavage. We demonstrate ATP coordination and conformational flexibility of the SF2-type helicase domain. Cas3 is specifically guided toward Cascade-bound target DNA by a PAM sequence, through physical interactions with both the nontarget substrate strand and the CasA protein. The sequence of recognition events ensures well-controlled DNA targeting and degradation of foreign DNA by Cascade and Cas3.
Assuntos
Actinomycetales/enzimologia , Proteínas Associadas a CRISPR/metabolismo , DNA Helicases/metabolismo , Actinomycetales/química , Actinomycetales/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Proteínas Associadas a CRISPR/química , Cristalografia por Raios X , DNA Helicases/química , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação ProteicaRESUMO
U-insertion/deletion RNA editing of mitochondrial mRNAs in trypanosome mitochondria is mediated by a core complex (RECC) containing around 16-20 proteins which is linked to several other multiprotein complexes by RNA. There are two known subcomplexes in the RECC: the REL1 subcomplex which contains the REL1 RNA ligase, the MP63 zinc finger-containing protein and the REX2 U-specific 3'-5' exonuclease; and the REL2 subcomplex which contains the REL2 RNA ligase, the RET2 3' TUTase and the MP81 zinc finger-containing protein. In this study we have affinity isolated recombinant TAP-tagged Leishmania major RET2 and Leishmania tarentolae MP63, REL1 and REL2 proteins after expression in baculovirus-infected insect cells. Recombinant MP63 protein was found to stimulate several in vitro activities of recombinant REL1; these activities include autoadenylation, bridged ligation and even pre-cleaved gRNA-mediated U-insertion editing with RET2 which is in the REL2 subcomplex. There was no effect of recombinant MP63 on similar REL2 ligation activities. The specificity for REL1 is consistent with MP63 being a component of the REL1 subcomplex. These results suggest that in vivo the interaction of MP63 with REL1 may play a role in regulating the overall activity of RNA editing.
Assuntos
Carbono-Oxigênio Ligases/metabolismo , Leishmania/metabolismo , Mitocôndrias/metabolismo , Proteínas de Protozoários/metabolismo , Edição de RNA , Proteínas de Ligação a RNA/metabolismo , Uridina/metabolismo , Animais , Baculoviridae , Vetores Genéticos , Leishmania/enzimologia , Mitocôndrias/enzimologia , Modelos Biológicos , Modelos Químicos , Mapeamento de Interação de Proteínas , Estrutura Quaternária de Proteína , Proteínas de Protozoários/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Dedos de ZincoRESUMO
Uridine (U)-insertion/deletion RNA editing in trypanosome mitochondria involves an initial cleavage of the preedited mRNA at specific sites determined by the annealing of partially complementary guide RNAs. An involvement of two RNase III-containing core editing complex (L-complex) proteins, MP90 (KREPB1) and MP61 (KREPB3) in, respectively, U-deletion and U-insertion editing, has been suggested, but these putative enzymes have not been characterized or expressed in active form. Recombinant MP90 proteins from Trypanosoma brucei and Leishmania major were expressed in insect cells and cytosol of Leishmania tarentolae, respectively. These proteins were active in specifically cleaving a model U-deletion site and not a U-insertion site. Deletion or mutation of the RNase III motif abolished this activity. Full-round guide RNA (gRNA)-mediated in vitro U-deletion editing was reconstituted by a mixture of recombinant MP90 and recombinant RNA editing exonuclease I from L. major, and recombinant RNA editing RNA ligase 1 from L. tarentolae. MP90 is designated REN1, for RNA-editing nuclease 1.
Assuntos
Proteínas de Protozoários/metabolismo , Edição de RNA , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Proteínas Recombinantes/metabolismo , Uridina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteína Catiônica de Eosinófilo/metabolismo , Regulação da Expressão Gênica , Leishmania/genética , Leishmania/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Proteínas de Protozoários/genética , RNA/genética , Interferência de RNA , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMO
Condensins are essential protein complexes critical for mitotic chromosome organization. Little is known about the function of condensins during interphase, particularly in mammalian cells. Here we report the interphase-specific interaction between condensin I and the DNA nick-sensor poly(ADP-ribose) polymerase 1 (PARP-1). We show that the association between condensin I, PARP-1, and the base excision repair (BER) factor XRCC1 increases dramatically upon single-strand break damage (SSB) induction. Damage-specific association of condensin I with the BER factors flap endonuclease 1 (FEN-1) and DNA polymerase delta/epsilon was also observed, suggesting that condensin I is recruited to interact with BER factors at damage sites. Consistent with this, DNA damage rapidly stimulates the chromatin association of PARP-1, condensin I, and XRCC1. Furthermore, depletion of condensin in vivo compromises SSB but not double-strand break (DSB) repair. Our results identify a SSB-specific response of condensin I through PARP-1 and demonstrate a role for condensin in SSB repair.
Assuntos
Adenosina Trifosfatases/fisiologia , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Complexos Multiproteicos/fisiologia , Poli(ADP-Ribose) Polimerases/genética , Adenosina Trifosfatases/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Transporte , Proteínas de Ciclo Celular , Linhagem Celular , Galinhas/genética , Cromatina , Proteínas Cromossômicas não Histona , DNA de Cadeia Simples , Células HeLa , Humanos , Interfase , Espectrometria de Massas , Camundongos/genética , Camundongos Knockout , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Proteínas Nucleares , Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/fisiologia , Transfecção , Proteína 1 Complementadora Cruzada de Reparo de Raio-X , Proteínas de XenopusRESUMO
Uridine insertion/deletion RNA editing in trypanosomatid mitochondria is a posttranscriptional RNA modification phenomenon required for translation of mitochondrial mRNAs. This process involves guide RNA-mediated cleavage at a specific site, insertion or deletion of Us from the 3' end of the 5' mRNA fragment, and ligation of the two mRNA fragments. The Leishmania major RNA ligase-containing complex protein 2 expressed in insect cells has a 3'-5' exoribonuclease activity and was therefore renamed RNA editing exonuclease 1 (REX1). Recombinant REX1 specifically trims 3' overhanging Us and stops at a duplex region. Evidence is presented that REX1 is responsible for deletion of the 3' overhanging Us from the bridged mRNA 5' cleavage fragment and that RNA editing ligase 1 is responsible for the ligation of the two mRNA cleavage fragments in U-deletion editing. The evidence involves both in vivo down-regulation of REX1 expression in Trypanosoma brucei by RNA interference and the reconstitution of precleaved U-deletion in vitro editing with only two recombinant enzymes: recombinant REX1 and recombinant RNA editing ligase 1.
Assuntos
Carbono-Oxigênio Ligases/fisiologia , Proteínas Mitocondriais/fisiologia , Edição de RNA , Trypanosoma brucei brucei/genética , Uridina/metabolismo , Animais , Proteínas Recombinantes/farmacologiaRESUMO
Smad proteins are critical intracellular mediators of the transforming growth factor-beta, bone morphogenic proteins (BMPs), and activin signaling. Upon ligand binding, the receptor-associated R-Smads are phosphorylated by the active type I receptor serine/threonine kinases. The phosphorylated R-Smads then form heteromeric complexes with Smad4, translocate into the nucleus, and interact with various transcription factors to regulate the expression of downstream genes. Interaction of Smad proteins with cellular partners in the cytoplasm and nucleus is a critical mechanism by which the activities and expression of the Smad proteins are modulated. Here we report a novel step of regulation of the R-Smad function at the inner nuclear membrane through a physical interaction between the integral inner nuclear membrane protein MAN1 and R-Smads. MAN1, through the RNA recognition motif, associates with R-Smads but not Smad4 at the inner nuclear membrane in a ligand-independent manner. Overexpression of MAN1 results in inhibition of R-Smad phosphorylation, heterodimerization with Smad4 and nuclear translocation, and repression of transcriptional activation of the TGFbeta, BMP2, and activin-responsive promoters. This repression of TGFbeta, BMP2, and activin signaling is dependent on the MAN1-Smad interaction because a point mutation that disrupts this interaction abolishes the transcriptional repression by MAN1. Thus, MAN1 represents a new class of R-Smad regulators and defines a previously unrecognized regulatory step at the nuclear periphery.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/metabolismo , Humanos , Proteínas de Membrana/genética , Membrana Nuclear/metabolismo , Proteínas Nucleares/genética , Fosforilação , Regiões Promotoras Genéticas , Transdução de Sinais , Proteínas SmadRESUMO
The X gene of hepatitis B virus (HBV) is one of the major factors in HBV-induced hepatocarcinogenesis and is essential for the establishment of productive HBV replication in vivo. Recent studies have shown that the X gene product targets mitochondria and induces calcium flux, thereby activating Ca(+)-dependent signal transduction pathways. However, regulatory mechanisms of X gene expression have remained unclear. Previous studies had localized a minimal promoter activity to a 21-bp GC-rich sequence located 130 bp upstream of the X protein coding region and showed that there was a cellular protein bound to this DNA. Interestingly, the 21-bp sequence identified as an X gene minimal promoter does not contain any previously identified core promoter elements, such as a TATA box. To better understand the mechanisms of transcriptional initiation of the X gene, we set out to biochemically purify the binding protein(s) for the 21-bp DNA. We report here the identification of the X gene minimal promoter-binding activity as nuclear respiratory factor 1 (NRF1), a previously known transcription factor that activates the majority of nucleus-encoded mitochondrial genes and various housekeeping genes. Primer extension analyses of the X mRNAs show that mutations at the binding site specifically inactivate transcription from this promoter and that a dominant-negative NRF1 mutant and short interfering RNAs inhibit transcription from this promoter. Therefore, NRF1 specifically binds the 21-bp minimal promoter and positively contributes to transcription of the X gene. Simultaneous activation of the X gene and mitochondrial genes by NRF1 may allow the X protein to target mitochondria most efficiently.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Vírus da Hepatite B/metabolismo , Regiões Promotoras Genéticas/fisiologia , Transativadores/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células HeLa , Vírus da Hepatite B/genética , Humanos , Dados de Sequência Molecular , Fator 1 Nuclear Respiratório , Fatores Nucleares Respiratórios , Regiões Promotoras Genéticas/genética , Transativadores/química , Transativadores/genética , Proteínas Virais Reguladoras e AcessóriasRESUMO
The human activator-recruited cofactor (ARC), a family of large transcriptional coactivator complexes related to the yeast Mediator, was recently identified based on functional association with the activation domains of multiple cellular and viral transcriptional activators, including the herpes simplex viral activator VP16, sterol regulatory element binding protein, and NF-kappaB. Here we describe the biochemical purification and cloning of the 92-kDa ARC/Mediator subunit, ARC92, that is specifically targeted by the activation domain of the VP16 transactivator. Affinity chromatography using the VP16 activation domain followed by peptide microsequencing led to the identification of ARC92 as a specific cellular interaction partner of the VP16 activation domain. ARC92 associates with the VP16 activation domain in vitro and in vivo, and the VP16 binding domain of ARC92 is a strong competitive inhibitor of Gal4-VP16 in vivo. Moreover, small interfering RNA-mediated knockdown of ARC92 in human cells results in selective inhibition of Gal4-VP16 gene activation. Taken together, our results suggest that ARC92 is a direct and specific target of the VP16 transactivator that serves in the context of the ARC/Mediator coactivator as an important transducer of transcription activating signals from the VP16 activation domain to the RNA polymerase II transcriptional machinery.
Assuntos
Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Núcleo Celular/metabolismo , Clonagem Molecular , Primers do DNA , DNA Complementar/genética , Células HeLa , Humanos , Complexo Mediador , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Polimerase II , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Transativadores/genética , Fatores de Transcrição/genética , Transcrição Gênica , Ativação Transcricional/genética , TransfecçãoRESUMO
Depletion of CD4(+) T cells is the hallmark of HIV infection and AIDS progression. In addition to the direct killing of the viral-infected cells, HIV infection also leads to increased apoptosis of predominantly uninfected bystander cells. This is mediated in part through the HIV-1 Tat protein, which is secreted by the infected cells and taken up by uninfected cells. Using an affinity-purification approach, a specific and direct interaction of Tat with tubulin and polymerized microtubules has been detected. This interaction does not affect the secretion and uptake of Tat, but is critical for Tat to induce apoptosis. Tat binds tubulin/microtubules through a four-amino-acid subdomain of its conserved core region, leading to the alteration of microtubule dynamics and activation of a mitochondria-dependent apoptotic pathway. Bim, a pro-apoptotic Bcl-2 relative and a transducer of death signals initiated by perturbation of microtubule dynamics, facilitates the Tat-induced apoptosis. Our findings reveal a strategy by which Tat induces apoptosis by targeting the microtubule network. Thus HIV-1 Tat joins a growing list of pathogen-derived proteins that target the cytoskeleton of host cells.
Assuntos
Apoptose , Proteínas de Transporte/metabolismo , Produtos do Gene tat/metabolismo , HIV-1/metabolismo , Proteínas de Membrana , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas , Animais , Proteínas Reguladoras de Apoptose , Proteína 11 Semelhante a Bcl-2 , Linfócitos T CD4-Positivos/metabolismo , Morte Celular , Linhagem Celular , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Dimerização , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Feminino , Citometria de Fluxo , Fase G1 , Glutationa Transferase/metabolismo , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Retroviridae/genética , Transfecção , Tubulina (Proteína)/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
The mammalian SWI/SNF-related complexes facilitate gene transcription by remodeling chromatin using the energy of ATP hydrolysis. The recruitment of these complexes to promoters remains poorly understood and may involve histone modifications or direct interactions with site-specific transcription factors or other cofactors. Here we report the isolation of two related but distinct cDNA clones, hOsa1 and hOsa2, that encode the largest subunits of human SWI/SNF. hOsa1 is identical to previously reported BAF250, and hOsa2 shares a high degree of sequence similarity with hOsa1. Mass spectrometric analysis, and immunoblotting with antibodies specific to hOsa1 or hOsa2 demonstrate the presence of both proteins in SWI/SNF-A but not in the related polybromo-BRG1-associated factors complex purified from HeLa cells. Co-precipitation studies indicate that hOsa1 and hOsa2 associate with BRG1 and hBRM through the C-terminal domain of hOsa. We define multiple domains within hBRM and BRG1 that interact with the hOsa C terminus. In cultured mammalian cells, hOsa1 and hOsa2 stimulate transcription by the glucocorticoid, estrogen, and androgen receptors. The glucocorticoid receptor-mediated activation is not observed with the C-terminal domain or with the hOsa2 polypeptide lacking the ARID DNA binding domain. These results suggest that hOsa1 and hOsa2 participate in promoting transcriptional activation by the steroid hormone receptors.
Assuntos
Proteínas Cromossômicas não Histona/química , Receptores de Esteroides/fisiologia , Fatores de Transcrição/química , Ativação Transcricional , Sequência de Aminoácidos , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/fisiologia , DNA Complementar/isolamento & purificação , Células HeLa , Humanos , Dados de Sequência Molecular , Subunidades Proteicas , Receptores Androgênicos/fisiologia , Receptores de Estrogênio/fisiologia , Receptores de Glucocorticoides/fisiologia , Proteínas Recombinantes/análise , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
Drosophila TATA-box-binding protein (TBP)-related factor 2 (TRF2) is a member of a family of TBP-related factors present in metazoan organisms. Recent evidence suggests that TRF2s are required for proper embryonic development and differentiation. However, true target promoters and the mechanisms by which TRF2 operates to control transcription remain elusive. Here we report the antibody affinity purification of a Drosophila TRF2-containing complex that contains components of the nucleosome remodelling factor (NURF) chromatin remodelling complex as well as the DNA replication-related element (DRE)-binding factor DREF. This latter finding led us to potential target genes containing TRF2-responsive promoters. We have used a combination of in vitro and in vivo assays to show that the DREF-containing TRF2 complex directs core promoter recognition of the proliferating cell nuclear antigen (PCNA) gene. We also identified additional TRF2-responsive target genes involved in DNA replication and cell proliferation. These data suggest that TRF2 functions as a core promoter-selectivity factor responsible for coordinating transcription of a subset of genes in Drosophila.
Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Drosophila/metabolismo , Regulação da Expressão Gênica , Regiões Promotoras Genéticas/genética , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Genes de Insetos/genética , Substâncias Macromoleculares , Ensaios de Proteção de Nucleases , Análise de Sequência com Séries de Oligonucleotídeos , Testes de Precipitina , Antígeno Nuclear de Célula em Proliferação/genética , Ligação Proteica , Interferência de RNA , Especificidade por Substrato , Proteína 2 de Ligação a Repetições Teloméricas/genética , Fatores de Transcrição/genética , Sítio de Iniciação de TranscriçãoRESUMO
DOCK180 protein plays a key role during development, cell motility, and phagocytosis. It forms a complex with another protein ELMO, and this complex acts as a guanine nucleotide exchange factor (GEF) for Rac. However, DOCK180-containing complexes have not been purified by unbiased biochemical approaches, and the nature and subcellular localization of these complexes remain unclear. Here, we show that a large fraction of endogenous DOCK180 is present as a 700kDa nuclear complex with ELMO proteins. In addition, this nuclear DOCK180/ELMO complex has functional Rac-GEF activity. Furthermore, endogenous DOCK180 could be found in complexes with different ELMO isoforms (ELMO1, 2 or 3) in different cell lines, depending on the ELMO isoforms expressed. These studies suggest that DOCK180 may associate with different ELMO proteins to form cell-type specific complexes and may have functions in both the nucleus and the cytoplasm.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Sítios de Ligação , Células CHO , Extratos Celulares/análise , Cricetinae , Cricetulus , Células HeLa , Humanos , Ligação Proteica , Isoformas de Proteínas/metabolismo , Distribuição TecidualRESUMO
There is considerable interest in the developmental, temporal and tissue-specific patterns of DNA replication in metazoans. Site-specific DNA replication at the chorion loci in Drosophila follicle cells leads to extensive gene amplification, and the organization of the cis-acting DNA elements that regulate this process may provide a model for how such regulation is achieved. Two elements important for amplification of the third chromosome chorion gene cluster, ACE3 and Ori-beta, are directly bound by Orc (origin recognition complex), and two-dimensional gel analysis has revealed that the primary origin used is Ori-beta (refs 7-9). Here we show that the Drosophila homologue of the Myb (Myeloblastosis) oncoprotein family is tightly associated with four additional proteins, and that the complex binds site-specifically to these regulatory DNA elements. Drosophila Myb is required in trans for gene amplification, showing that a Myb protein is directly involved in DNA replication. A Drosophila Myb binding site, as well as the binding site for another Myb complex member (p120), is necessary in cis for replication of reporter transgenes. Chromatin immunoprecipitation experiments localize both proteins to the chorion loci in vivo. These data provide evidence that specific protein complexes bound to replication enhancer elements work together with the general replication machinery for site-specific origin utilization during replication.