RESUMO
Lipoprotein lipase (LPL) plays a central role in triglyceride (TG) metabolism. By catalyzing the hydrolysis of TGs present in TG-rich lipoproteins (TRLs), LPL facilitates TG utilization and regulates circulating TG and TRL concentrations. Until very recently, structural information for LPL was limited to homology models, presumably due to the propensity of LPL to unfold and aggregate. By coexpressing LPL with a soluble variant of its accessory protein glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) and with its chaperone protein lipase maturation factor 1 (LMF1), we obtained a stable and homogenous LPL/GPIHBP1 complex that was suitable for structure determination. We report here X-ray crystal structures of human LPL in complex with human GPIHBP1 at 2.5-3.0 Å resolution, including a structure with a novel inhibitor bound to LPL. Binding of the inhibitor resulted in ordering of the LPL lid and lipid-binding regions and thus enabled determination of the first crystal structure of LPL that includes these important regions of the protein. It was assumed for many years that LPL was only active as a homodimer. The structures and additional biochemical data reported here are consistent with a new report that LPL, in complex with GPIHBP1, can be active as a monomeric 1:1 complex. The crystal structures illuminate the structural basis for LPL-mediated TRL lipolysis as well as LPL stabilization and transport by GPIHBP1.
Assuntos
Lipase Lipoproteica/química , Lipase Lipoproteica/metabolismo , Receptores de Lipoproteínas/química , Receptores de Lipoproteínas/metabolismo , Células HEK293 , Humanos , Hidrólise , Metabolismo dos Lipídeos/fisiologia , Lipólise/fisiologia , Lipoproteínas/metabolismo , Triglicerídeos/metabolismoRESUMO
Lipoprotein lipase (LPL) is central to triglyceride metabolism. Severely compromised LPL activity causes familial chylomicronemia syndrome (FCS), which is associated with very high plasma triglyceride levels and increased risk of life-threatening pancreatitis. Currently, no approved pharmacological intervention can acutely lower plasma triglycerides in FCS. Low yield, high aggregation, and poor stability of recombinant LPL have thus far prevented development of enzyme replacement therapy. Recently, we showed that LPL monomers form 1:1 complexes with the LPL transporter glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) and solved the structure of the complex. In the present work, we further characterized the monomeric LPL/GPIHBP1 complex and its derivative, the LPL-GPIHBP1 fusion protein, with the goal of contributing to the development of an LPL enzyme replacement therapy. Fusion of LPL to GPIHBP1 increased yields of recombinant LPL, prevented LPL aggregation, stabilized LPL against spontaneous inactivation, and made it resistant to inactivation by the LPL antagonists angiopoietin-like protein 3 (ANGPTL3) or ANGPTL4. The high stability of the fusion protein enabled us to identify LPL amino acids that interact with ANGPTL4. Additionally, the LPL-GPIHBP1 fusion protein exhibited high enzyme activity in in vitro assays. Importantly, both intravenous and subcutaneous administrations of the fusion protein lowered triglycerides in several mouse strains without causing adverse effects. These results indicate that the LPL-GPIHBP1 fusion protein has potential for use as a therapeutic for managing FCS.
Assuntos
Lipase Lipoproteica/metabolismo , Receptores de Lipoproteínas/metabolismo , Triglicerídeos/sangue , Sequência de Aminoácidos , Proteína 3 Semelhante a Angiopoietina , Proteína 4 Semelhante a Angiopoietina/química , Proteína 4 Semelhante a Angiopoietina/metabolismo , Proteínas Semelhantes a Angiopoietina/química , Proteínas Semelhantes a Angiopoietina/metabolismo , Animais , Sítios de Ligação , Modelos Animais de Doenças , Terapia de Reposição de Enzimas , Humanos , Hiperlipoproteinemia Tipo I/tratamento farmacológico , Hiperlipoproteinemia Tipo I/patologia , Infusões Subcutâneas , Lipase Lipoproteica/química , Lipase Lipoproteica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Agregados Proteicos/efeitos dos fármacos , Estabilidade Proteica , Receptores de Lipoproteínas/química , Receptores de Lipoproteínas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/uso terapêuticoRESUMO
Completion of DNA replication after replication stress depends on PCNA, which undergoes monoubiquitination to stimulate direct bypass of DNA lesions by specialized DNA polymerases or is polyubiquitinated to promote recombination-dependent DNA synthesis across DNA lesions by template switching mechanisms. Here we report that the ZRANB3 translocase, a SNF2 family member related to the SIOD disorder SMARCAL1 protein, is recruited by polyubiquitinated PCNA to promote fork restart following replication arrest. ZRANB3 depletion in mammalian cells results in an increased frequency of sister chromatid exchange and DNA damage sensitivity after treatment with agents that cause replication stress. Using in vitro biochemical assays, we show that recombinant ZRANB3 remodels DNA structures mimicking stalled replication forks and disassembles recombination intermediates. We therefore propose that ZRANB3 maintains genomic stability at stalled or collapsed replication forks by facilitating fork restart and limiting inappropriate recombination that could occur during template switching events.
Assuntos
DNA Helicases/metabolismo , Replicação do DNA/fisiologia , Instabilidade Genômica/fisiologia , Poliubiquitina/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Estresse Fisiológico/genética , Sequência de Aminoácidos , Linhagem Celular Tumoral , Dano ao DNA/fisiologia , DNA Helicases/genética , Proteínas de Fluorescência Verde/genética , Humanos , Dados de Sequência Molecular , Osteossarcoma , Ligação Proteica/fisiologia , Recombinação Genética/fisiologia , Troca de Cromátide Irmã/fisiologia , Ubiquitinação/fisiologiaRESUMO
Repair of dsDNA breaks requires processing to produce 3'-terminated ssDNA. We biochemically reconstituted DNA end resection using purified human proteins: Bloom helicase (BLM); DNA2 helicase/nuclease; Exonuclease 1 (EXO1); the complex comprising MRE11, RAD50, and NBS1 (MRN); and Replication protein A (RPA). Resection occurs via two routes. In one, BLM and DNA2 physically and specifically interact to resect DNA in a process that is ATP-dependent and requires BLM helicase and DNA2 nuclease functions. RPA is essential for both DNA unwinding by BLM and enforcing 5' â 3' resection polarity by DNA2. MRN accelerates processing by recruiting BLM to the end. In the other, EXO1 resects the DNA and is stimulated by BLM, MRN, and RPA. BLM increases the affinity of EXO1 for ends, and MRN recruits and enhances the processivity of EXO1. Our results establish two of the core machineries that initiate recombinational DNA repair in human cells.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Hidrolases Anidrido Ácido , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Quebras de DNA de Cadeia Simples , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Helicases/fisiologia , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/fisiologia , Humanos , Técnicas In Vitro , Proteína Homóloga a MRE11 , Modelos Biológicos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Ligação Proteica/fisiologia , RecQ Helicases/genética , RecQ Helicases/metabolismo , RecQ Helicases/fisiologia , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Proteína de Replicação A/fisiologiaRESUMO
The Saccharomyces cerevisiae Dmc1 and Tid1 proteins are required for the pairing of homologous chromosomes during meiotic recombination. This pairing is the precursor to the formation of crossovers between homologs, an event that is necessary for the accurate segregation of chromosomes. Failure to form crossovers can have serious consequences and may lead to chromosomal imbalance. Dmc1, a meiosis-specific paralog of Rad51, mediates the pairing of homologous chromosomes. Tid1, a Rad54 paralog, although not meiosis-specific, interacts with Dmc1 and promotes crossover formation between homologs. In this study, we show that purified Dmc1 and Tid1 interact physically and functionally. Dmc1 forms stable nucleoprotein filaments that can mediate DNA strand invasion. Tid1 stimulates Dmc1-mediated formation of joint molecules. Under conditions optimal for Dmc1 reactions, Rad51 is specifically stimulated by Rad54, establishing that Dmc1-Tid1 and Rad51-Rad54 function as specific pairs. Physical interaction studies show that specificity in function is not dictated by direct interactions between the proteins. Our data are consistent with the hypothesis that Rad51-Rad54 function together to promote intersister DNA strand exchange, whereas Dmc1-Tid1 tilt the bias toward interhomolog DNA strand exchange.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromossomos Fúngicos/metabolismo , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , DNA Topoisomerases/metabolismo , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Rad51 Recombinase/metabolismo , Recombinação Genética/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Cromossomos Fúngicos/genética , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , DNA Topoisomerases/genética , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Meiose/fisiologia , Rad51 Recombinase/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Saccharomyces cerevisiae Rad52 performs multiple functions during the recombinational repair of double-stranded DNA (dsDNA) breaks (DSBs). It mediates assembly of Rad51 onto single-stranded DNA (ssDNA) that is complexed with replication protein A (RPA); the resulting nucleoprotein filament pairs with homologous dsDNA to form joint molecules. Rad52 also catalyzes the annealing of complementary strands of ssDNA, even when they are complexed with RPA. Both Rad51 and Rad52 can be envisioned to promote "second-end capture," a step that pairs the ssDNA generated by processing of the second end of a DSB to the joint molecule formed by invasion of the target dsDNA by the first processed end. Here, we show that Rad52 promotes annealing of complementary ssDNA that is complexed with RPA to the displaced strand of a joint molecule, to form a complement-stabilized joint molecule. RecO, a prokaryotic homolog of Rad52, cannot form complement-stabilized joint molecules with RPA-ssDNA complexes, nor can Rad52 promote second-end capture when the ssDNA is bound with either human RPA or the prokaryotic ssDNA-binding protein, SSB, indicating a species-specific process. We conclude that Rad52 participates in second-end capture by annealing a resected DNA break, complexed with RPA, to the joint molecule product of single-end invasion event. These studies support a role for Rad52-promoted annealing in the formation of Holliday junctions in DSB repair.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , DNA/genética , Plasmídeos/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Humanos , Ligação Proteica , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidade por SubstratoRESUMO
The error-free repair of double-stranded DNA breaks by homologous recombination requires processing of broken ends. These processed ends are substrates for assembly of DNA strand exchange proteins that mediate DNA strand invasion. Here, we establish that human BLM helicase, a member of the RecQ family, stimulates the nucleolytic activity of human exonuclease 1 (hExo1), a 5'-->3' double-stranded DNA exonuclease. The stimulation is specific because other RecQ homologs fail to stimulate hExo1. Stimulation of DNA resection by hExo1 is independent of BLM helicase activity and is, instead, mediated by an interaction between the 2 proteins. Finally, we show that DNA ends resected by hExo1 and BLM are used by human Rad51, but not its yeast or bacterial counterparts, to promote homologous DNA pairing. This in vitro system recapitulates initial steps of homologous recombination and provides biochemical evidence for a role of BLM and Exo1 in the initiation of recombinational DNA repair.
Assuntos
DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Adenosina Trifosfatases/metabolismo , DNA/química , Quebras de DNA de Cadeia Dupla , DNA Helicases/química , DNA Helicases/genética , Enzimas Reparadoras do DNA/química , Exodesoxirribonucleases/química , Humanos , Rad51 Recombinase/metabolismo , RecQ Helicases/metabolismo , Recombinação Genética , Proteína de Replicação A/metabolismoRESUMO
ICP8, the herpes simplex virus type-1 encoded single-strand DNA (ssDNA)-binding protein, promotes the assimilation of a single-stranded DNA molecule into a homologous duplex plasmid resulting in the formation of a displacement loop. Here we examine the mechanism of this process. In contrast to the RecA-type recombinases that catalyze strand invasion via an active search for homology, ICP8 acts by a salt-dependent strand annealing mechanism. The active species in this reaction is a ssDNA:ICP8 nucleoprotein filament. There appears to be no requirement for ICP8 to interact with the acceptor DNA. At higher concentrations, ICP8 promotes the reverse reaction, presumably owing to its helix destabilizing activity. ICP8-mediated strand assimilation imparts single-stranded character onto the acceptor DNA, consistent with the formation of a displacement loop. These data suggest that the recombination activity of ICP8 is similar to the mechanism of eukaryotic Rad52.
Assuntos
DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Virais/metabolismo , DNA de Cadeia Simples/química , Modelos Químicos , Conformação de Ácido Nucleico , Nucleoproteínas/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Recombinases Rec A/metabolismo , TemperaturaRESUMO
The Saccharomyces cerevisiae Tid1 protein is important for the recombinational repair of double-stranded DNA breaks during meiosis. Tid1 is a member of Swi2/Snf2 family of chromatin remodeling proteins and shares homology with Rad54. Members of this family hydrolyze ATP and promote 1) chromatin remodeling, 2) DNA topology alterations, and 3) displacement of proteins from DNA. All of these activities are presumed to require translocation of the protein on DNA. Here we use single-molecule visualization to provide direct evidence for the ability of Tid1 to translocate on DNA. Tid1 translocation is ATP-dependent, and the velocities are broadly distributed, with the average being 84 +/- 39 base pairs/s. Translocation is processive, with the average molecule traveling approximately 10,000 base pairs before pausing or dissociating. Many molecules display simple monotonic unidirectional translocation, but the majority display complex translocation behavior comprising intermittent pauses, direction reversals, and velocity changes. Finally, we demonstrate that translocation by Tid1 on DNA can result in disruption of three-stranded DNA structures. The ability of Tid1 translocation to clear DNA of proteins and to migrate recombination intermediates may be of critical importance for DNA repair and chromosome dynamics.
Assuntos
Trifosfato de Adenosina/química , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , DNA/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Adenosina Trifosfatases , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Cromossomos Fúngicos/química , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , DNA/genética , DNA/metabolismo , DNA Helicases , Enzimas Reparadoras do DNA , DNA Topoisomerases , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Hidrólise , Ligação Proteica/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Recombination-dependent replication is an integral part of the process by which double-strand DNA breaks are repaired to maintain genome integrity. It also serves as a means to replicate genomic termini. We reported previously on the reconstitution of a recombination-dependent replication system using purified herpes simplex virus type 1 proteins (Nimonkar A. V., and Boehmer, P. E. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10201-10206). In this system, homologous pairing by the viral single-strand DNA-binding protein (ICP8) is coupled to DNA synthesis by the viral DNA polymerase and helicase-primase in the presence of a DNA-relaxing enzyme. Here we show that DNA synthesis in this system is dependent on the viral polymerase processivity factor (UL42). Moreover, although DNA synthesis is strictly dependent on topoisomerase I, it is only stimulated by the viral helicase in a manner that requires the helicase-loading protein (UL8). Furthermore, we have examined the dependence of DNA synthesis in the viral system on species-specific protein-protein interactions. Optimal DNA synthesis was observed with the herpes simplex virus type 1 replication proteins, ICP8, DNA polymerase (UL30/UL42), and helicase-primase (UL5/UL52/UL8). Interestingly, substitution of each component with functional homologues from other systems for the most part did not drastically impede DNA synthesis. In contrast, recombination-dependent replication promoted by the bacteriophage T7 replisome was disrupted by substitution with the replication proteins from herpes simplex virus type 1. These results show that although DNA synthesis performed by the T7 replisome is dependent on cognate protein-protein interactions, such interactions are less important in the herpes simplex virus replisome.
Assuntos
Herpesvirus Humano 1/genética , Recombinação Genética , Replicação Viral , Bacteriófago T7/metabolismo , DNA/química , DNA Helicases/química , DNA Helicases/fisiologia , DNA Primase , Proteínas de Ligação a DNA , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Relação Dose-Resposta a Droga , Exodesoxirribonucleases/metabolismo , Herpesvirus Humano 1/enzimologia , Oligonucleotídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Fatores de Tempo , Proteínas Virais/metabolismo , Proteínas Virais/fisiologiaRESUMO
The repair of double-strand DNA breaks by homologous recombination is essential for the maintenance of genome stability. In herpes simplex virus 1, double-strand DNA breaks may arise as a consequence of replication fork collapse at sites of oxidative damage, which is known to be induced upon viral infection. Double-strand DNA breaks are also generated by cleavage of viral a sequences by endonuclease G during genome isomerization. We have reconstituted a system using purified proteins in which strand invasion is coupled with DNA synthesis. In this system, the viral single-strand DNA-binding protein promotes assimilation of single-stranded DNA into a homologous supercoiled plasmid, resulting in the formation of a displacement loop. The 3' terminus of the invading DNA serves as a primer for long-chain DNA synthesis promoted by the viral DNA replication proteins, including the polymerase and helicase-primase. Efficient extension of the invading primer also requires a DNA-relaxing enzyme (eukaryotic topoisomerase I or DNA gyrase). The viral polymerase by itself is insufficient for DNA synthesis, and a DNA-relaxing enzyme cannot substitute for the viral helicase-primase. The viral single-strand DNA-binding protein, in addition to its role in the invasion process, is also required for long-chain DNA synthesis. Form X, a topologically distinct, positively supercoiled form of displacement-loop, does not serve as a template for DNA synthesis. These observations support a model in which recombination and replication contribute toward maintaining viral genomic stability by repairing double-strand breaks. They also account for the extensive branching observed during viral replication in vivo.
Assuntos
DNA Viral/biossíntese , Herpesvirus Humano 1/genética , Recombinação Genética , Replicação do DNA , Proteínas de Ligação a DNA , Moldes Genéticos , Proteínas Virais/fisiologiaRESUMO
The genome of herpes simplex virus type-1 undergoes a high frequency of homologous recombination in the absence of a virus-encoded RecA-type protein. We hypothesized that viral homologous recombination is mediated by the combined action of the viral single strand DNA-binding protein (ICP8) and helicase-primase. Our results show that ICP8 catalyzes the formation of recombination intermediates (joint molecules) between circular single-stranded acceptor and linear duplex donor DNA. Joint molecules formed by invasion of a 3'-terminal strand displaces the non-complementary 5'-terminal strand, thereby creating a loading site for the helicase-primase. Helicase-primase acts on these joint molecules to promote ATP-dependent branch migration. Finally, we have reconstituted strand exchange by the synchronous action of ICP8 and helicase-primase. Based on these data, we present a recombination mechanism for a eukaryotic DNA virus in which a single strand DNA-binding protein and helicase cooperate to promote homologous pairing and branch migration.
Assuntos
DNA Helicases/metabolismo , DNA Primase/metabolismo , Herpesvirus Humano 1/metabolismo , Proteínas Virais/metabolismo , Catálise , Replicação do DNA , DNA Viral/biossíntese , DNA Viral/metabolismo , Proteínas de Ligação a DNA , Recombinação GenéticaRESUMO
ICP8, the herpes simplex virus type-1 single-strand DNA-binding protein, was recently shown to promote strand exchange in conjunction with the viral replicative helicase (Nimonkar, A. V., and Boehmer, P. E. (2002) J. Biol. Chem. 277, 15182-15189). Here we show that ICP8 also catalyzes strand invasion in an ATP-independent manner. Thus, ICP8 promotes the assimilation of a single-stranded donor molecule into a homologous plasmid, resulting in the formation of a displacement loop. Invasion of a homologous duplex by single-stranded DNA requires homology at either 3' or 5' end of the invading strand. The reaction is dependent on the free energy of supercoiling and alters the topology of the acceptor plasmid. Hence, strand invasion products formed by ICP8 are resistant to the action of restriction endonucleases that cleave outside of the area of pairing. The ability to catalyze strand invasion is a novel activity of ICP8 and the first demonstration of a eukaryotic viral single-strand DNA-binding protein to promote this reaction. In this regard ICP8 is functionally similar to the prototypical prokaryotic recombinase RecA and its eukaryotic homologs. This strand invasion activity of ICP8 coupled with DNA synthesis may explain the high prevalence of branched DNA structures during viral replication.
Assuntos
Ácidos Nucleicos/química , Proteínas Virais/química , Trifosfato de Adenosina/metabolismo , Catálise , Proteínas de Ligação a DNA , Relação Dose-Resposta a Droga , Eletroforese em Gel de Ágar , Endonucleases/metabolismo , Etídio/farmacologia , Plasmídeos/metabolismo , Recombinases Rec A/metabolismo , Proteínas Virais/metabolismoRESUMO
Herpesviruses are large double stranded DNA animal viruses with the distinguishing ability to establish latent, life-long infections. To date, eight human herpesviruses that exhibit distinct biological and corresponding pathological/clinical properties have been identified. During their life cycles, herpesviruses execute an intricate chain of events geared towards optimizing their replication. This sets an interesting paradigm to study fundamental biological processes. This review summarizes recent developments in herpesvirus research with emphasis on genome transactions, particularly with respect to the prototypic herpes simplex virus type-1.