Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 61
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nucleic Acids Res ; 49(20): 11653-11665, 2021 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-34718732

RESUMO

The CST complex (CTC1-STN1-TEN1) has been shown to inhibit telomerase extension of the G-strand of telomeres and facilitate the switch to C-strand synthesis by DNA polymerase alpha-primase (pol α-primase). Recently the structure of human CST was solved by cryo-EM, allowing the design of mutant proteins defective in telomeric ssDNA binding and prompting the reexamination of CST inhibition of telomerase. The previous proposal that human CST inhibits telomerase by sequestration of the DNA primer was tested with a series of DNA-binding mutants of CST and modeled by a competitive binding simulation. The DNA-binding mutants had substantially reduced ability to inhibit telomerase, as predicted from their reduced affinity for telomeric DNA. These results provide strong support for the previous primer sequestration model. We then tested whether addition of CST to an ongoing processive telomerase reaction would terminate DNA extension. Pulse-chase telomerase reactions with addition of either wild-type CST or DNA-binding mutants showed that CST has no detectable ability to terminate ongoing telomerase extension in vitro. The same lack of inhibition was observed with or without pol α-primase bound to CST. These results suggest how the switch from telomerase extension to C-strand synthesis may occur.

2.
PLoS One ; 16(2): e0237956, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606679

RESUMO

Cyp33 is an essential human cyclophilin prolyl isomerase that plays myriad roles in splicing and chromatin remodeling. In addition to a canonical cyclophilin (Cyp) domain, Cyp33 contains an RNA-recognition motif (RRM) domain, and RNA-binding triggers proline isomerase activity. One prominent role for Cyp33 is through a direct interaction with the mixed lineage leukemia protein 1 (MLL1, also known as KMT2A) complex, which is a histone methyltransferase that serves as a global regulator of human transcription. MLL activity is regulated by Cyp33, which isomerizes a key proline in the linker between the PHD3 and Bromo domains of MLL1, acting as a switch between gene activation and repression. The direct interaction between MLL1 and Cyp33 is critical, as deletion of the MLL1-PHD3 domain responsible for this interaction results in oncogenesis. The Cyp33 RRM is central to these activities, as it binds both the PHD3 domain and RNA. To better understand how RNA binding drives the action of Cyp33, we performed RNA-SELEX against full-length Cyp33 accompanied by deep sequencing. We have identified an enriched Cyp33 binding motif (AAUAAUAA) broadly represented in the cellular RNA pool as well as tightly binding RNA aptamers with affinities comparable and competitive with the Cyp33 MLL1-PHD3 interaction. RNA binding extends beyond the canonical RRM domain, but not to the Cyp domain, suggesting an indirect mechanism of interaction. NMR chemical shift mapping confirms an overlapping, but not identical, interface on Cyp33 for RNA and PHD3 binding. This finding suggests RNA can disrupt the gene repressive Cyp33-MLL1 complex providing another layer of regulation for chromatin remodeling by MLL1.


Assuntos
Ciclofilinas/metabolismo , Regulação da Expressão Gênica/genética , Histona-Lisina N-Metiltransferase/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Sequência de Aminoácidos/genética , Sítios de Ligação/genética , Ciclofilinas/genética , Proteínas de Ligação a DNA/genética , Expressão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Humanos , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Nucleares/genética , Motivos de Nucleotídeos/genética , Ligação Proteica/genética , RNA/metabolismo , Motivo de Reconhecimento de RNA/genética , Técnica de Seleção de Aptâmeros/métodos , Fatores de Transcrição/metabolismo
3.
Nucleic Acids Res ; 48(16): 9320-9335, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32813011

RESUMO

Heterogeneous nuclear ribonuclear protein K (hnRNPK) is an abundant RNA-binding protein crucial for a wide variety of biological processes. While its binding preference for multi-cytosine-patch (C-patch) containing RNA is well documented, examination of binding to known cellular targets that contain C-patches reveals an unexpected breadth of binding affinities. Analysis of in-cell crosslinking data reinforces the notion that simple C-patch preference is not fully predictive of hnRNPK localization within transcripts. The individual RNA-binding domains of hnRNPK work together to interact with RNA tightly, with the KH3 domain being neither necessary nor sufficient for binding. Rather, the RG/RGG domain is implicated in providing essential contributions to RNA-binding, but not DNA-binding, affinity. hnRNPK is essential for X chromosome inactivation, where it interacts with Xist RNA specifically through the Xist B-repeat region. We use this interaction with an RNA motif derived from this B-repeat region to determine the RNA-structure dependence of C-patch recognition. While the location preferences of hnRNPK for C-patches are conformationally restricted within the hairpin, these structural constraints are relieved in the absence of RNA secondary structure. Together, these results illustrate how this multi-domain protein's ability to accommodate and yet discriminate between diverse cellular RNAs allows for its broad cellular functions.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Motivos de Nucleotídeos/genética , RNA Longo não Codificante/genética , Ribonucleoproteínas/genética , Animais , Pareamento de Bases/genética , Núcleo Celular/genética , Feminino , Humanos , Proteínas de Ligação a RNA/genética , Inativação do Cromossomo X/genética
4.
Science ; 368(6495): 1081-1085, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32499435

RESUMO

The CTC1-STN1-TEN1 (CST) complex is essential for telomere maintenance and resolution of stalled replication forks genome-wide. Here, we report the 3.0-angstrom cryo-electron microscopy structure of human CST bound to telomeric single-stranded DNA (ssDNA), which assembles as a decameric supercomplex. The atomic model of the 134-kilodalton CTC1 subunit, built almost entirely de novo, reveals the overall architecture of CST and the DNA-binding anchor site. The carboxyl-terminal domain of STN1 interacts with CTC1 at two separate docking sites, allowing allosteric mediation of CST decamer assembly. Furthermore, ssDNA appears to staple two monomers to nucleate decamer assembly. CTC1 has stronger structural similarity to Replication Protein A than the expected similarity to yeast Cdc13. The decameric structure suggests that CST can organize ssDNA analogously to the nucleosome's organization of double-stranded DNA.


Assuntos
Complexos Multiproteicos/química , Homeostase do Telômero , Proteínas de Ligação a Telômeros/química , Telômero/química , Microscopia Crioeletrônica , DNA de Cadeia Simples/química , Células HEK293 , Humanos , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Proteína de Replicação A/química
5.
Elife ; 92020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32379046

RESUMO

The Xist lncRNA mediates X chromosome inactivation (XCI). Here we show that Spen, an Xist-binding repressor protein essential for XCI , binds to ancient retroviral RNA, performing a surveillance role to recruit chromatin silencing machinery to these parasitic loci. Spen loss activates a subset of endogenous retroviral (ERV) elements in mouse embryonic stem cells, with gain of chromatin accessibility, active histone modifications, and ERV RNA transcription. Spen binds directly to ERV RNAs that show structural similarity to the A-repeat of Xist, a region critical for Xist-mediated gene silencing. ERV RNA and Xist A-repeat bind the RRM domains of Spen in a competitive manner. Insertion of an ERV into an A-repeat deficient Xist rescues binding of Xist RNA to Spen and results in strictly local gene silencing in cis. These results suggest that Xist may coopt transposable element RNA-protein interactions to repurpose powerful antiviral chromatin silencing machinery for sex chromosome dosage compensation.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Retrovirus Endógenos/genética , Células-Tronco Embrionárias Murinas/virologia , RNA Longo não Codificante/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Inativação do Cromossomo X , Cromossomo X , Animais , Sítios de Ligação , Linhagem Celular , Proteínas de Ligação a DNA/genética , Compensação de Dosagem (Genética) , Retrovirus Endógenos/metabolismo , Feminino , Interações Hospedeiro-Patógeno , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica , RNA Longo não Codificante/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genética
6.
Nat Commun ; 11(1): 1805, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286318

RESUMO

Certain transcription factors are proposed to form functional interactions with RNA to facilitate proper regulation of gene expression. Sox2, a transcription factor critical for maintenance of pluripotency and neurogenesis, has been found associated with several lncRNAs, although it is unknown whether these interactions are direct or via other proteins. Here we demonstrate that human Sox2 interacts directly with one of these lncRNAs with high affinity through its HMG DNA-binding domain in vitro. These interactions are primarily with double-stranded RNA in a non-sequence specific fashion, mediated by a similar but not identical interaction surface. We further determined that Sox2 directly binds RNA in mouse embryonic stem cells by UV-cross-linked immunoprecipitation of Sox2 and more than a thousand Sox2-RNA interactions in vivo were identified using fRIP-seq. Together, these data reveal that Sox2 employs a high-affinity/low-specificity paradigm for RNA binding in vitro and in vivo.


Assuntos
RNA/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Sequência de Bases , Ligação Competitiva , DNA/metabolismo , Masculino , Camundongos , Modelos Moleculares , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica , Domínios Proteicos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fatores de Transcrição SOXB1/química , Deleção de Sequência , Eletricidade Estática
7.
Biochemistry ; 58(51): 5107-5111, 2019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31829559

RESUMO

Poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2, respectively), upon binding damaged DNA, become activated to add long chains of poly(ADP-ribose) (PAR) to themselves and other nuclear proteins. This activation is an essential part of the DNA damage response. The PAR modifications recruit the DNA repair machinery to sites of DNA damage and result in base excision and single-strand break repair, homologous recombination, nucleotide excision repair, and alternative nonhomologous end joining. More recently, both PARP1 and PARP2 have been shown to bind to or be activated by RNA, a property that could interfere with the function of PARP1 and PARP2 in the response to DNA damage or lead to necrosis by depletion of cellular NAD+. We have quantitatively evaluated the in vitro binding of a variety of RNAs to PARP1 and PARP2 and queried the ability of these RNAs to switch on enzymatic activity. We find that while both proteins bind RNAs without specificity toward sequence or structure, their interaction with RNA does not lead to auto-PARylation. Thus, although PARP1 and PARP2 are promiscuous with respect to activation by DNA, they both demonstrate exquisite selectivity against activation by RNA.


Assuntos
Poli(ADP-Ribose) Polimerase-1/metabolismo , RNA/metabolismo , Modelos Moleculares , Poli(ADP-Ribose) Polimerase-1/química , Ligação Proteica , Domínios Proteicos
8.
Nucleic Acids Res ; 47(15): 8180-8192, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31147715

RESUMO

The glucocorticoid receptor (GR) binds the noncoding RNA Gas5 via its DNA-binding domain (DBD) with functional implications in pro-apoptosis signaling. Here, we report a comprehensive in vitro binding study where we have determined that GR-DBD is a robust structure-specific RNA-binding domain. GR-DBD binds to a diverse range of RNA hairpin motifs, both synthetic and biologically derived, with apparent mid-nanomolar affinity while discriminating against uniform dsRNA. As opposed to dimeric recognition of dsDNA, GR-DBD binds to RNA as a monomer and confers high affinity primarily through electrostatic contacts. GR-DBD adopts a discrete RNA-bound state, as assessed by NMR, distinct from both free and DNA-bound. NMR and alanine mutagenesis suggest a heightened involvement of the C-terminal α-helix of the GR-DBD in RNA-binding. RNA competes for binding with dsDNA and occurs in a similar affinity range as dimer binding to the canonical DNA element. Given the prevalence of RNA hairpins within the transcriptome, our findings strongly suggest that many RNAs have potential to impact GR biology.


Assuntos
Proteínas de Ligação a DNA/química , DNA/química , RNA/química , Receptores de Glucocorticoides/química , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , RNA/metabolismo , Receptores de Glucocorticoides/metabolismo
9.
Methods Mol Biol ; 1855: 341-354, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30426430

RESUMO

Measuring protein/DNA interactions that have apparent binding affinity constants in the low-picomolar range presents a unique experimental challenge. To probe the sequence specificity of telomere binding proteins, our laboratory has developed an electrophoretic mobility shift assay protocol that allows for the routine measurement of K D,app values in the 1-20 pM range. Here, we describe the protocol and highlight the particular considerations that should be made to successfully and reproducibly measure high-affinity interactions between proteins and single-stranded DNA.


Assuntos
DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Resinas Acrílicas/química , Animais , Humanos , Ligação Proteica
10.
Proc Natl Acad Sci U S A ; 115(41): 10315-10320, 2018 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-30249661

RESUMO

ssDNA, which is involved in numerous aspects of chromosome biology, is managed by a suite of proteins with tailored activities. The majority of these proteins bind ssDNA indiscriminately, exhibiting little apparent sequence preference. However, there are several notable exceptions, including the Saccharomyces cerevisiae Cdc13 protein, which is vital for yeast telomere maintenance. Cdc13 is one of the tightest known binders of ssDNA and is specific for G-rich telomeric sequences. To investigate how these two different biochemical features, affinity and specificity, contribute to function, we created an unbiased panel of alanine mutations across the Cdc13 DNA-binding interface, including several aromatic amino acids that play critical roles in binding activity. A subset of mutant proteins exhibited significant loss in affinity in vitro that, as expected, conferred a profound loss of viability in vivo. Unexpectedly, a second category of mutant proteins displayed an increase in specificity, manifested as an inability to accommodate changes in ssDNA sequence. Yeast strains with specificity-enhanced mutations displayed a gradient of viability in vivo that paralleled the loss in sequence tolerance in vitro, arguing that binding specificity can be fine-tuned to ensure optimal function. We propose that DNA binding by Cdc13 employs a highly cooperative interface whereby sequence diversity is accommodated through plastic binding modes. This suggests that sequence specificity is not a binary choice but rather is a continuum. Even in proteins that are thought to be specific nucleic acid binders, sequence tolerance through the utilization of multiple binding modes may be a broader phenomenon than previously appreciated.


Assuntos
DNA de Cadeia Simples/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Sítios de Ligação , Mutagênese Sítio-Dirigida , Mutação , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/química
11.
Structure ; 26(5): 722-733.e2, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29681468

RESUMO

Pot1 is the shelterin component responsible for the protection of the single-stranded DNA (ssDNA) overhang at telomeres in nearly all eukaryotic organisms. The C-terminal domain of the DNA-binding domain, Pot1pC, exhibits non-specific ssDNA recognition, achieved through thermodynamically equivalent alternative binding conformations. Given this flexibility, it is unclear how specificity for ssDNA over RNA, an activity required for biological function, is achieved. Examination of the ribose-position specificity of Pot1pC shows that ssDNA specificity is additive but not uniformly distributed across the ligand. High-resolution structures of several Pot1pC complexes with RNA-DNA chimeric ligands reveal Pot1pC discriminates against RNA by utilizing non-compensatory binding modes that feature significant rearrangement of the binding interface. These alternative conformations, accessed through both ligand and protein flexibility, recover much, but not all, of the binding energy, leading to the observed reduction in affinities. These findings suggest that intermolecular interfaces are remarkably sophisticated in their tuning of specificity toward flexible ligands.


Assuntos
DNA de Cadeia Simples/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/metabolismo , Sítios de Ligação , Ligantes , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , RNA/metabolismo , Ribose/metabolismo
12.
Proteins ; 86(2): 164-176, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29127727

RESUMO

SH2B1 is a multidomain protein that serves as a key adaptor to regulate numerous cellular events, such as insulin, leptin, and growth hormone signaling pathways. Many of these protein-protein interactions are mediated by the SH2 domain of SH2B1, which recognizes ligands containing a phosphorylated tyrosine (pY), including peptides derived from janus kinase 2, insulin receptor, and insulin receptor substrate-1 and -2. Specificity for the SH2 domain of SH2B1 is conferred in these ligands either by a hydrophobic or an acidic side chain at the +3 position C-terminal to the pY. This specificity for chemically disparate species suggests that SH2B1 relies on distinct thermodynamic or structural mechanisms to bind to peptides. Using binding and structural strategies, we have identified unique thermodynamic signatures for each peptide binding mode, and several SH2B1 residues, including K575 and R578, that play distinct roles in peptide binding. The high-resolution structure of the SH2 domain of SH2B1 further reveals conformationally plastic protein loops that may contribute to the ability of the protein to recognize dissimilar ligands. Together, numerous hydrophobic and electrostatic interactions, in addition to backbone conformational flexibility, permit the recognition of diverse peptides by SH2B1. An understanding of this expanded peptide recognition will allow for the identification of novel physiologically relevant SH2B1/peptide interactions, which can contribute to the design of obesity and diabetes pharmaceuticals to target the ligand-binding interface of SH2B1 with high specificity.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeos/metabolismo , Domínios de Homologia de src , Proteínas Adaptadoras de Transdução de Sinal/química , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Peptídeos/química , Ligação Proteica , Conformação Proteica , Mapas de Interação de Proteínas , Eletricidade Estática , Termodinâmica
13.
Biochemistry ; 56(32): 4210-4218, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28726394

RESUMO

The human CST (CTC1-STN1-TEN1) heterotrimeric complex plays roles in both telomere maintenance and DNA replication through its ability to interact with single-stranded DNA (ssDNA) of a variety of sequences. The precise sequence specificity required to execute these functions is unknown. Telomere-binding proteins have been shown to specifically recognize key telomeric sequence motifs within ssDNA while accommodating nonspecifically recognized sequences through conformationally plastic interfaces. To better understand the role CST plays in these processes, we have produced a highly purified heterotrimer and elucidated the sequence requirements for CST recognition of ssDNA in vitro. CST discriminates against random sequence and binds a minimal ssDNA comprised of three repeats of telomeric sequence. Replacement of individual nucleotides with their complement reveals that guanines are specifically recognized in a largely additive fashion and that specificity is distributed uniformly throughout the ligand. Unexpectedly, adenosines are also well tolerated at these sites, but cytosines are disfavored. Furthermore, sequences unrelated to the telomere repeat, yet still G-rich, bind CST well. Thus, CST is not inherently telomere-specific, but rather is a G-rich sequence binder. This biochemical activity is reminiscent of the yeast t-RPA and Tetrahymena thermophila CST complexes and is consistent with roles at G-rich sites throughout the genome.


Assuntos
Complexos Multiproteicos/química , Motivos de Nucleotídeos , Proteínas de Ligação a Telômeros/química , Telômero/química , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo
14.
Biochemistry ; 56(16): 2225-2237, 2017 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-28376302

RESUMO

SH2 domains recognize phosphotyrosine (pY)-containing peptide ligands and play key roles in the regulation of receptor tyrosine kinase pathways. Each SH2 domain has individualized specificity, encoded in the amino acids neighboring the pY, for defined targets that convey their distinct functions. The C-terminal SH2 domain (PLCC) of the phospholipase C-γ1 full-length protein (PLCγ1) typically binds peptides containing small and hydrophobic amino acids adjacent to the pY, including a peptide derived from platelet-derived growth factor receptor B (PDGFRB) and an intraprotein recognition site (Y783 of PLCγ1) involved in the regulation of the protein's lipase activity. Remarkably, PLCC also recognizes unexpected peptides containing amino acids with polar or bulky side chains that deviate from this pattern. This versatility in recognition specificity may allow PLCγ1 to participate in diverse, previously unrecognized, signaling pathways in response to binding chemically dissimilar partners. We have used structural approaches, including nuclear magnetic resonance and X-ray crystallography, to elucidate the mechanisms of noncognate peptide binding to PLCC by ligands derived from receptor tyrosine kinase ErbB2 and from the insulin receptor. The high-resolution peptide-bound structures reveal that PLCC has a relatively static backbone but contains a chemically rich protein surface comprised of a combination of hydrophobic pockets and amino acids with charged side chains. We demonstrate that this expansive and chemically diverse PLCC interface, in addition to peptide conformational plasticity, permits PLCC to recognize specific noncognate peptide ligands with multimodal specificity.


Assuntos
Peptídeos/metabolismo , Fosfolipase C gama/metabolismo , Domínios de Homologia de src , Sítios de Ligação , Calorimetria , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Fosfolipase C gama/química , Conformação Proteica
15.
Methods Mol Biol ; 1555: 269-290, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28092038

RESUMO

Heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) experiments offer a rapid and high resolution approach to gaining binding and conformational insights into a protein-peptide interaction. By tracking 1H and 15N chemical shift changes over the course of a peptide titration into isotopically labeled protein, amide NH pairs of amino acids whose chemical environment changes upon peptide binding can be identified. When mapped onto a structure of the protein, this approach can identify the peptide-binding interface or regions undergoing conformation changes within a protein upon ligand binding. Monitoring NMR chemical shift changes can also serve as a screening technique to identify novel interaction partners for a protein or to determine the binding affinity of a weak protein-peptide interaction. Here, we describe the application of NMR chemical shift mapping to the study of peptide binding to the C-terminal SH2 domain of PLCγ1.


Assuntos
Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Domínios e Motivos de Interação entre Proteínas , Domínios de Homologia de src , Algoritmos , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Expressão Gênica , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade
16.
Methods Mol Biol ; 1555: 291-305, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28092039

RESUMO

Isothermal titration calorimetry (ITC) has emerged as a leading approach in the characterization of protein/ligand interactions. This technique measures the heat change of a system upon binding of a ligand to a biomolecule, and thereby requires no immobilization, intrinsic fluorescence, or labeling of any kind of either species. If properly designed, a single experiment can not only measure the binding affinity, but also determine additional binding and thermodynamic parameters, including the enthalpy, entropy, and the stoichiometry of the interaction. Here, we describe the protocol for the collection of calorimetric data for the binding of peptides to SH2 protein domains.


Assuntos
Calorimetria/métodos , Ligantes , Domínios de Homologia de src , Clonagem Molecular , Entropia , Expressão Gênica , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Proteínas/química , Proteínas/metabolismo , Proteínas Recombinantes de Fusão , Termodinâmica
17.
Biochemistry ; 55(38): 5326-40, 2016 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-27575340

RESUMO

Telomeres terminate nearly exclusively in single-stranded DNA (ssDNA) overhangs comprised of the G-rich 3' end. This overhang varies widely in length from species to species, ranging from just a few bases to several hundred nucleotides. These overhangs are not merely a remnant of DNA replication but rather are the result of complex further processing. Proper management of the telomeric overhang is required both to deter the action of the DNA damage machinery and to present the ends properly to the replicative enzyme telomerase. This Current Topic addresses the biochemical and structural features used by the proteins that manage these variable telomeric overhangs. The Pot1 protein tightly binds the single-stranded overhang, preventing DNA damage sensors from binding. Pot1 also orchestrates the access of telomerase to that same substrate. The remarkable plasticity of the binding interface exhibited by the Schizosaccharomyces pombe Pot1 provides mechanistic insight into how these roles may be accomplished, and disease-associated mutations clustered around the DNA-binding interface in the hPOT1 highlight the importance of this function. The budding yeast Cdc13-Stn1-Ten1, a telomeric RPA complex closely associated with telomere function, also interacts with ssDNA in a fashion that allows degenerate sequences to be recognized. A related human complex composed of hCTC1, hSTN1, and hTEN1 has recently emerged with links to both telomere maintenance and general DNA replication and also exhibits mutations associated with telomere pathologies. Overall, these sequence-specific ssDNA binders exhibit a range of recognition properties that allow them to perform their unique biological functions.


Assuntos
DNA de Cadeia Simples/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/química , Telômero
18.
Cell Rep ; 15(10): 2170-2184, 2016 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-27239034

RESUMO

Genome sequencing studies have revealed a number of cancer-associated mutations in the telomere-binding factor POT1. Here, we show that when combined with p53 deficiency, depletion of murine POT1a in common lymphoid progenitor cells fosters genetic instability, accelerates the onset, and increases the severity of T cell lymphomas. In parallel, we examined human and mouse cells carrying POT1 mutations found in cutaneous T cell lymphoma (CTCL) patients. Inhibition of POT1 activates ATR-dependent DNA damage signaling and induces telomere fragility, replication fork stalling, and telomere elongation. Our data suggest that these phenotypes are linked to impaired CST (CTC1-STN1-TEN1) function at telomeres. Lastly, we show that proliferation of cancer cells lacking POT1 is enabled by the attenuation of the ATR kinase pathway. These results uncover a role for defective telomere replication during tumorigenesis.


Assuntos
Carcinogênese/metabolismo , Carcinogênese/patologia , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Estresse Fisiológico , Telômero/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Predisposição Genética para Doença , Instabilidade Genômica , Células Progenitoras Linfoides/metabolismo , Linfoma Cutâneo de Células T/genética , Linfoma Cutâneo de Células T/imunologia , Linfoma Cutâneo de Células T/patologia , Camundongos , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Proteínas de Ligação a Telômeros , Timo/patologia , Proteína Supressora de Tumor p53/metabolismo
19.
Structure ; 22(9): 1223-1224, 2014 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-25185825

RESUMO

By merging recent experimental and computational methodology advances, resolution-adapted structural recombination Rosetta has emerged as a powerful strategy for solving the structure of traditionally challenging targets. In this issue of Structure, Sgourakis and colleagues solve the structure of one such target, the immunoevasin protein m04, using this approach.


Assuntos
Proteínas de Transporte/química , Glicoproteínas/química , Proteínas Virais/química
20.
Nucleic Acids Res ; 42(15): 9656-65, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25074378

RESUMO

Telomere protection and length regulation are important processes for aging, cancer and several other diseases. At the heart of these processes lies the single-stranded DNA (ssDNA)-binding protein Pot1, a component of the telomere maintenance complex shelterin, which is present in species ranging from fission yeast to humans. Pot1 contains a dual OB-fold DNA-binding domain (DBD) that fully confers its high affinity for telomeric ssDNA. Studies of S. pombe Pot1-DBD and its individual OB-fold domains revealed a complex non-additive behavior of the two OB-folds in the context of the complete Pot1 protein. This behavior includes the use of multiple distinct binding modes and an ability to form higher order complexes. Here we use NMR and biochemical techniques to investigate the structural features of the complete Pot1-DBD. These experiments reveal one binding mode characterized by only subtle alternations to the individual OB-fold subdomain structures, resulting in an inaccessible 3' end of the ssDNA. The second binding mode, which has equivalent affinity, interacts differently with the 3' end, rendering it available for interaction with other proteins. These findings suggest a structural switch that contributes to telomere end-protection and length regulation.


Assuntos
DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/metabolismo , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...