RESUMO
The conserved Notch signaling pathway plays crucial roles in developing and self-renewing tissues. Notch is activated upon ligand-induced conformation change of the Notch negative regulatory region (NRR) unmasking a key proteolytic site (S2) and facilitating downstream events. Thus far, the molecular mechanism of this signal activation is not defined. However, strong indirect evidence favors a model whereby transendocytosis of the Notch extracellular domain, in tight association with ligand into the ligand-bearing cell, exerts a force on the NRR to drive the required structure change. Here, we demonstrate that force applied to the human Notch2 NRR can indeed expose the S2 site and, crucially, allow cleavage by the metalloprotease TACE (TNF-alpha-converting enzyme). Molecular insight into this process is achieved using atomic force microscopy and molecular dynamics simulations on the human Notch2 NRR. The data show near-sequential unfolding of its constituent LNR (Lin12-Notch repeat) and HD (heterodimerization) domains, at forces similar to those observed for other protein domains with a load-bearing role. Exposure of the S2 site is the first force "barrier" on the unfolding pathway, occurring prior to unfolding of any domain, and achieved via removal of the LNRAB linker region from the HD domain. Metal ions increase the resistance of the Notch2 NRR to forced unfolding, their removal clearly facilitating unfolding at lower forces. The results provide direct demonstration of force-mediated exposure and cleavage of the Notch S2 site and thus firmly establish the feasibility of a mechanotransduction mechanism for ligand-induced Notch activation.
Assuntos
Proteínas ADAM/metabolismo , Receptor Notch2/química , Receptor Notch2/metabolismo , Sequências Reguladoras de Ácido Nucleico , Proteína ADAM17 , Sequência de Aminoácidos , Sítios de Ligação/genética , Western Blotting , Humanos , Ligantes , Microscopia de Força Atômica , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , Desdobramento de Proteína , Proteólise , Receptor Notch2/genética , Transdução de SinaisRESUMO
Ribonuclease MRP is an endonuclease, related to RNase P, which functions in eukaryotic pre-rRNA processing. In Saccharomyces cerevisiae, RNase MRP comprises an RNA subunit and ten proteins. To improve our understanding of subunit roles and enzyme architecture, we have examined protein-protein and protein-RNA interactions in vitro, complementing existing yeast two-hybrid data. In total, 31 direct protein-protein interactions were identified, each protein interacting with at least three others. Furthermore, seven proteins self-interact, four strongly, pointing to subunit multiplicity in the holoenzyme. Six protein subunits interact directly with MRP RNA and four with pre-rRNA. A comparative analysis with existing data for the yeast and human RNase P/MRP systems enables confident identification of Pop1p, Pop4p and Rpp1p as subunits that lie at the enzyme core, with probable addition of Pop5p and Pop3p. Rmp1p is confirmed as an integral subunit, presumably associating preferentially with RNase MRP, rather than RNase P, via interactions with Snm1p and MRP RNA. Snm1p and Rmp1p may act together to assist enzyme specificity, though roles in substrate binding are also indicated for Pop4p and Pop6p. The results provide further evidence of a conserved eukaryotic RNase P/MRP architecture and provide a strong basis for studies of enzyme assembly and subunit function.
Assuntos
Endorribonucleases/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Endorribonucleases/isolamento & purificação , Humanos , Mapeamento de Interação de Proteínas , Subunidades Proteicas/isolamento & purificação , Precursores de RNA/metabolismo , RNA Fúngico/metabolismo , RNA Ribossômico/metabolismo , Ribonuclease P/metabolismo , Ribonucleoproteínas/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/isolamento & purificaçãoRESUMO
In vitro transcripts of bacteriophage RNA polymerases (RNAPs), such as T7 RNAP, often suffer from a considerable degree of 3'-end heterogeneity and, with certain promoter sequences, 5'-end heterogeneity. For some applications, this transcript heterogeneity poses a significant problem. A potential solution is to incorporate ribozymes into the transcripts at the 5'- and/or 3'-end of the target RNA sequence. This approach has been used quite widely but has required the generation of new transcription vectors or PCR-derived templates for each new RNA to be studied. To overcome this limitation, we have created two general plasmids for producing homogeneous RNA transcripts: one encodes a 3'- hepatitis delta virus (HDV) ribozyme and the other, used in combination with a two-step PCR, allows the production of double [5'-hammerhead (HH) and 3'-HDV] ribozyme constructs. A choice of cloning and run-off transcription linearisation restriction enzyme sites ensures that virtually any RNA sequence can be cloned and transcribed from these plasmids. For all the RNA sequences tested, good yields of transcript were obtained. These plasmids provide the tools for the simple, rapid creation of new RNA-coding plasmids to produce milligram quantities of homogeneous in vitro transcripts for all applications.
Assuntos
Vetores Genéticos , Plasmídeos , RNA/biossíntese , Transcrição Gênica , Sequência de Bases , Clonagem Molecular , Vírus Delta da Hepatite/enzimologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA/química , RNA Catalítico/genética , Moldes GenéticosRESUMO
RNase MRP is a ribonucleoprotein endoribonuclease involved in eukaryotic pre-rRNA processing. The enzyme possesses a putatively catalytic RNA subunit, structurally related to that of RNase P. A thorough structure analysis of Saccharomyces cerevisiae MRP RNA, entailing enzymatic and chemical probing, mutagenesis and thermal melting, identifies a previously unrecognised stem that occupies a position equivalent to the P7 stem of RNase P. Inclusion of this P7-like stem confers on yeast MRP RNA a greater degree of similarity to the core RNase P RNA structure than that described previously and better delimits domain 2, the proposed specificity domain. The additional stem is created by participation of a conserved sequence element (ymCR-II) in a long-range base-pairing interaction. There is potential for this base-pairing throughout the known yeast MRP RNA sequences. Formation of a P7-like stem is not required, however, for the pre-rRNA processing or essential function of RNase MRP. Mutants that can base-pair are nonetheless detrimental to RNase MRP function, indicating that the stem will form in vivo but that only the wild-type pairing is accommodated. Although the alternative MRP RNA structure described is clearly not part of the active RNase MRP enzyme, it would be the more stable structure in the absence of protein subunits and the probability that it represents a valid intermediate species in the process of yeast RNase MRP assembly is discussed.
Assuntos
Pareamento de Bases/genética , Sequência Conservada/genética , Endorribonucleases/genética , Conformação de Ácido Nucleico , RNA Fúngico/genética , Saccharomyces cerevisiae/enzimologia , Sequência de Bases , Sítios de Ligação , Endorribonucleases/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Subunidades Proteicas , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Catalítico/química , RNA Catalítico/genética , RNA Fúngico/química , TermodinâmicaRESUMO
The Notch signalling pathway is fundamental to cell differentiation in developing and self-renewing tissues. Notch is activated upon ligand-induced conformational change of the Notch negative regulatory region (NRR), unmasking a key proteolytic site (S2) and facilitating downstream events. The favoured model requires endocytosis of a tightly bound ligand to transmit force to the NRR region, sufficient to cause a structural change that exposes the S2 site. We have previously shown, using atomic force microscopy and molecular dynamics simulations, that application of force to the N-terminus of the Notch2 NRR facilitates metalloprotease cleavage at an early stage in the unfolding process. Here, mutations are made within the heterodimerization (HD) domain of the NRR that are known to cause constitutive activation of Notch1 whilst having no effect on the chemical stability of Notch2. Comparison of the mechanical stability and simulated forced unfolding of recombinant Notch2 NRR proteins demonstrates a reduced stability following mutation and identifies two critical structural elements of the NRR in its response to force - the linker region between Lin12-Notch repeats LNRA and LNRB and the α3 helix within the HD domain - both of which mask the S2 cleavage site prior to Notch activation. In two mutated proteins, the LNRC:HD domain interaction is also reduced in stability. The observed changes to mechanical stability following these HD domain mutations highlight key regions of the Notch2 NRR that are important for mechanical, but not chemical, stability. This research could also help determine the fundamental differences in the NRRs of Notch1 and Notch2.
RESUMO
The use of in vitro transcribed RNA is often limited by sequence constraints at the 5'-end and the problem of transcript heterogeneity which can occur at both the 5'- and 3'-ends. This chapter describes the use of cis-acting ribozymes, 5'-end hammerhead (HH) and 3'-end hepatitis delta virus (HDV), for direct transcriptional processing to yield target RNAs with precisely defined ends. The method is focused on the use of the pRZ and p2RZ plasmids that are designed to simplify the production of such dual ribozyme templates. These plasmids each bear a 3'-HDV modified with a unique restriction site that allows the ribozyme to remain on the plasmid and, therefore, be omitted from the cloning procedure. The additional steps required to design a unique hammerhead ribozyme tailored to the 5'-end of each target RNA are detailed. In most cases, a transcriptional template bearing a 5'-HH ribozyme and a 3'-HDV ribozyme can be achieved by cloning a single PCR product into either the pRZ or p2RZ vector. Protocols for optimization of transcription yields from these templates and the isolation of the homogeneous target RNA are also described.
Assuntos
Técnicas Genéticas , Vírus Delta da Hepatite/enzimologia , RNA Catalítico/metabolismo , Sequência de Bases , Clonagem Molecular , RNA Mensageiro/biossíntese , RNA Mensageiro/química , RNA Mensageiro/genética , Transcrição GênicaRESUMO
Zona occludens (ZO) proteins are molecular scaffolds localized to cell junctions, which regulate epithelial integrity in mammals. Using newly generated null alleles, we demonstrate that polychaetoid (pyd), the unique Drosophila melanogaster ZO homologue, regulates accumulation of adherens junction-localized receptors, such as Notch, although it is dispensable for epithelial polarization. Pyd positively regulates Notch signaling during sensory organ development but acts negatively on Notch to restrict the ovary germline stem cell niche. In both contexts, we identify a core antagonistic interaction between Pyd and the WW domain E3 ubiquitin ligase Su(dx). Pyd binds Su(dx) directly, in part through a noncanonical WW-binding motif. Pyd also restricts epithelial wing cell numbers to control adult wing shape, a function associated with the FERM protein Expanded and independent of Su(dx). As both Su(dx) and Expanded regulate trafficking, we propose that a conserved role of ZO proteins is to coordinate receptor trafficking and signaling with junctional organization.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Homologia de Sequência de Aminoácidos , Ubiquitina-Proteína Ligases/metabolismo , Junções Aderentes/metabolismo , Alelos , Animais , Drosophila melanogaster/citologia , Drosophila melanogaster/ultraestrutura , Feminino , Proteínas de Membrana/química , Mutação/genética , Ovário/citologia , Fosfoproteínas/química , Prolina/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Notch/metabolismo , Órgãos dos Sentidos/citologia , Órgãos dos Sentidos/embriologia , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismo , Relação Estrutura-Atividade , Proteínas de Junções Íntimas , Asas de Animais/anatomia & histologia , Asas de Animais/citologia , Asas de Animais/ultraestrutura , Proteína da Zônula de Oclusão-1RESUMO
A new NMR approach is presented for observing in vitro multicomponent protein-protein-ligand(s) interactions, which should help to understand how cellular networks of protein interactions operate on a molecular level and how they can be controlled with drugs. The method uniquely allows at least two polypeptide components of the mixture to be simultaneously closely monitored in a single sample, without increased signal overlap, and can be used to study complex (e.g., sequential, competitive, cooperative, allosteric, induced, etc.) binding events, witnessed by two polypeptides independently. One polypeptide is uniformly labeled with 15N and another with 15N and 13C. The 1H-15N correlation spectra are recorded for each of these molecules separately, discriminated on the basis of the type of 13C'/12C' atom attached to the amide group nitrogen. Any changes to the state of the two differently isotopically labeled molecules will be reported individually by fingerprint signals from amide groups, e.g., as unlabeled ligands are added. To our knowledge, no other technique currently exists which can monitor complex binding events in similar detail. The proposed method can be combined easily with traditional protein NMR techniques and incorporated in a variety of applications.
Assuntos
Proteínas/química , Simulação por Computador , Isótopos/química , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Sensibilidade e Especificidade , TitulometriaRESUMO
WW domains target proline-tyrosine (PY) motifs and frequently function as tandem pairs. When studied in isolation, single WW domains are notably promiscuous and regulatory mechanisms are undoubtedly required to ensure selective interactions. Here, we show that the fourth WW domain (WW4) of Suppressor of Deltex, a modular Nedd4-like protein that down-regulates the Notch receptor, is the primary mediator of a direct interaction with a Notch-PY motif. A natural Trp to Phe substitution in WW4 reduces its affinity for general PY sequences and enhances selective interaction with the Notch-PY motif via compensatory specificity-determining interactions with PY-flanking residues. When WW4 is paired with WW3, domain-domain association, impeding proper folding, competes with Notch-PY binding to WW4. This novel mode of autoinhibition is relieved by binding of another ligand to WW3. Such cooperativity may facilitate the transient regulatory interactions observed in vivo between Su(dx) and Notch in the endocytic pathway. The highly conserved tandem arrangement of WW domains in Nedd4 proteins, and similar arrangements in more diverse proteins, suggests domain-domain communication may be integral to regulation of their associated cellular activities.
Assuntos
Proteínas de Drosophila/química , Drosophila melanogaster/química , Proteínas de Membrana/química , Modelos Moleculares , Ubiquitina-Proteína Ligases/química , Motivos de Aminoácidos/fisiologia , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Endocitose/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ubiquitina-Proteína Ligases Nedd4 , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
RNase MRP is a ribonucleoprotein endoribonuclease involved in eukaryotic pre-rRNA processing. The enzyme possesses an RNA subunit, structurally related to that of RNase P RNA, that is thought to be catalytic. RNase MRP RNA sequences from Saccharomycetaceae species are structurally well defined through detailed phylogenetic and structural analysis. In contrast, higher eukaryote MRP RNA structure models are based on comparative sequence analysis of only five sequences and limited probing data. Detailed structural analysis of the Homo sapiens MRP RNA, entailing enzymatic and chemical probing, is reported. The data are consistent with the phylogenetic secondary structure model and demonstrate unequivocally that higher eukaryote MRP RNA structure differs significantly from that reported for Saccharomycetaceae species. Neither model can account for all of the known MRP RNAs and we thus propose the evolution of at least two subsets of RNase MRP secondary structure, differing predominantly in the predicted specificity domain.
Assuntos
Endorribonucleases/química , Conformação de Ácido Nucleico , RNA/química , Saccharomycetales/genética , Algoritmos , Animais , Sequência de Bases , Catálise , Evolução Molecular , Humanos , Dados de Sequência Molecular , Filogenia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomycetales/metabolismo , Homologia de Sequência do Ácido Nucleico , Especificidade por Substrato , Transcrição GênicaRESUMO
A key ion-dependent folding unit within the hepatitis C IRES comprises the IIIef junction and pseudoknot. This region is also important in recruitment of the 40S ribosomal subunit. Here, circular dichroism is used to study the influence of metal ions on the structure and stability of this region. Comparison of the thermal stability of an IRES fragment encompassing subdomains IIIe/f and IV (named 3EF4) with that of a larger fragment also possessing subdomain IIId (3DEF4) indicates an additional stabilizing effect of Mg(2+) ions on the latter fragment. Magnesium and potassium ions stabilize both fragments through nonspecific counterion effects. The additional effect of magnesium on 3DEF4, observed in the absence or presence of 100 mM KCl, is attributed to a nonspecific but high-affinity site for metal ions created by a region of unusual high charge density. Subdomain IIId presumably participates in tertiary packing interactions that provide such a site. Viomycin binds to the full-length IRES and RNA fragments with K(d) values of 25-55 microM. Interestingly, viomycin binding to the two fragments is affected differently by Mg(2+); noncompetitive inhibition of binding to 3DEF4 is observed, whereas binding to 3EF4 is not impaired. Formation of a Mg(2+)-stabilized tertiary fold, involving subdomain IIId, may thereby hinder viomycin binding to 3DEF4 indirectly. Mutational and deletion studies locate viomycin binding within subdomains IIIe/f rather than within the pseudoknot. In pseudoknot mutants, Mg(2+) ions have different effects on viomycin binding and thermal stability, suggesting altered tertiary interactions involving subdomain IIId.
Assuntos
Antibacterianos/química , Hepacivirus/química , Magnésio/química , Ribossomos/química , Viomicina/química , Sítios de Ligação/genética , Cálcio/química , Cátions Bivalentes/química , Dicroísmo Circular , Cobalto/química , Eletroforese em Gel de Poliacrilamida , Hepacivirus/genética , Cloreto de Magnésio/química , Manganês/química , Modelos Moleculares , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , RNA Viral/química , RNA Viral/genética , Ribossomos/genética , Deleção de SequênciaRESUMO
WW domains mediate protein recognition, usually though binding to proline-rich sequences. In many proteins, WW domains occur in tandem arrays. Whether or how individual domains within such arrays cooperate to recognize biological partners is, as yet, poorly characterized. An important question is whether functional diversity of different WW domain proteins is reflected in the structural organization and ligand interaction mechanisms of their multiple domains. We have determined the solution structure and dynamics of a pair of WW domains (WW3-4) from a Drosophila Nedd4 family protein called Suppressor of deltex (Su(dx)), a regulator of Notch receptor signaling. We find that the binding of a type 1 PPPY ligand to WW3 stabilizes the structure with effects propagating to the WW4 domain, a domain that is not active for ligand binding. Both WW domains adopt the characteristic triple-stranded beta-sheet structure, and significantly, this is the first example of a WW domain structure to include a domain (WW4) lacking the second conserved Trp (replaced by Phe). The domains are connected by a flexible linker, which allows a hinge-like motion of domains that may be important for the recognition of functionally relevant targets. Our results contrast markedly with those of the only previously determined three-dimensional structure of tandem WW domains, that of the rigidly oriented WW domain pair from the RNA-splicing factor Prp40. Our data illustrate that arrays of WW domains can exhibit a variety of higher order structures and ligand interaction mechanisms.