RESUMO
Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally distinct classes of chaperones promote de novo folding, suggesting that their activities are coordinated at the ribosome. We used biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We found that chaperone binding is disfavored close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor recognizes compact folding intermediates that expose an extensive unfolded surface, and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates and recruits DnaK to sequence-diverse solvent-accessible sites. Neither Trigger factor, DnaJ, nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to protect incipient structure in the nascent polypeptide well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70 , Biossíntese de Proteínas , Dobramento de Proteína , Ribossomos , Ribossomos/metabolismo , Ribossomos/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP40/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Ligação Proteica , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Modelos Moleculares , Conformação Proteica , Peptidilprolil IsomeraseRESUMO
Regulated protein phosphorylation controls most cellular processes. The protein phosphatase PP1 is the catalytic subunit of many holoenzymes that dephosphorylate serine/threonine residues. How these enzymes recruit their substrates is largely unknown. Here, we integrated diverse approaches to elucidate how the PP1 non-catalytic subunit PPP1R15B (R15B) captures its full trimeric eIF2 substrate. We found that the substrate-recruitment module of R15B is largely disordered with three short helical elements, H1, H2, and H3. H1 and H2 form a clamp that grasps the substrate in a region remote from the phosphorylated residue. A homozygous N423D variant, adjacent to H1, reducing substrate binding and dephosphorylation was discovered in a rare syndrome with microcephaly, developmental delay, and intellectual disability. These findings explain how R15B captures its 125 kDa substrate by binding the far end of the complex relative to the phosphosite to present it for dephosphorylation by PP1, a paradigm of broad relevance.
Assuntos
Domínio Catalítico , Fator de Iniciação 2 em Eucariotos , Proteína Fosfatase 1 , Humanos , Fosforilação , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismoRESUMO
Defects in organellar acidification indicate compromised or infected compartments. Recruitment of the autophagy-related ATG16L1 complex to pathologically neutralized organelles targets ubiquitin-like ATG8 molecules to perturbed membranes. How this process is coupled to proton gradient disruption is unclear. Here, we reveal that the V1H subunit of the vacuolar ATPase (V-ATPase) proton pump binds directly to ATG16L1. The V1H/ATG16L1 interaction only occurs within fully assembled V-ATPases, allowing ATG16L1 recruitment to be coupled to increased V-ATPase assembly following organelle neutralization. Cells lacking V1H fail to target ATG8s during influenza infection or after activation of the immune receptor stimulator of interferon genes (STING). We identify a loop within V1H that mediates ATG16L1 binding. A neuronal V1H isoform lacks this loop and is associated with attenuated ATG8 targeting in response to ionophores in primary murine and human iPSC-derived neurons. Thus, V1H controls ATG16L1 recruitment following proton gradient dissipation, suggesting that the V-ATPase acts as a cell-intrinsic damage sensor.
Assuntos
Proteínas Relacionadas à Autofagia , ATPases Vacuolares Próton-Translocadoras , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Humanos , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Animais , Camundongos , Ligação Proteica , Neurônios/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Autofagia , Células HEK293 , Células-Tronco Pluripotentes Induzidas/metabolismo , Influenza Humana/virologia , Influenza Humana/metabolismo , Influenza Humana/genética , Camundongos Endogâmicos C57BL , Transdução de Sinais , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Camundongos KnockoutRESUMO
Modular SCF (SKP1-CUL1-Fbox) ubiquitin E3 ligases orchestrate multiple cellular pathways in eukaryotes. Their variable SKP1-Fbox substrate receptor (SR) modules enable regulated substrate recruitment and subsequent proteasomal degradation. CAND proteins are essential for the efficient and timely exchange of SRs. To gain structural understanding of the underlying molecular mechanism, we reconstituted a human CAND1-driven exchange reaction of substrate-bound SCF alongside its co-E3 ligase DCNL1 and visualized it by cryo-EM. We describe high-resolution structural intermediates, including a ternary CAND1-SCF complex, as well as conformational and compositional intermediates representing SR- or CAND1-dissociation. We describe in molecular detail how CAND1-induced conformational changes in CUL1/RBX1 provide an optimized DCNL1-binding site and reveal an unexpected dual role for DCNL1 in CAND1-SCF dynamics. Moreover, a partially dissociated CAND1-SCF conformation accommodates cullin neddylation, leading to CAND1 displacement. Our structural findings, together with functional biochemical assays, help formulate a detailed model for CAND-SCF regulation.
Assuntos
Proteínas Culina , Proteínas Ligases SKP Culina F-Box , Humanos , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas Culina/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Transporte/metabolismoRESUMO
ATG9A and ATG2A are essential core members of the autophagy machinery. ATG9A is a lipid scramblase that allows equilibration of lipids across a membrane bilayer, whereas ATG2A facilitates lipid flow between tethered membranes. Although both have been functionally linked during the formation of autophagosomes, the molecular details and consequences of their interaction remain unclear. By combining data from peptide arrays, crosslinking, and hydrogen-deuterium exchange mass spectrometry together with cryoelectron microscopy, we propose a molecular model of the ATG9A-2A complex. Using this integrative structure modeling approach, we identify several interfaces mediating ATG9A-2A interaction that would allow a direct transfer of lipids from ATG2A into the lipid-binding perpendicular branch of ATG9A. Mutational analyses combined with functional activity assays demonstrate their importance for autophagy, thereby shedding light on this protein complex at the heart of autophagy.
Assuntos
Autofagossomos , Autofagia , Microscopia Crioeletrônica , Bioensaio , LipídeosRESUMO
Maintenance of epigenetic integrity relies on coordinated recycling and partitioning of parental histones and deposition of newly synthesized histones during DNA replication. This process depends upon a poorly characterized network of histone chaperones, remodelers, and binding proteins. Here we implicate the POLE3-POLE4 subcomplex of the leading-strand polymerase, Polε, in replication-coupled nucleosome assembly through its ability to selectively bind to histones H3-H4. Using hydrogen/deuterium exchange mass spectrometry and physical mapping, we define minimal domains necessary for interaction between POLE3-POLE4 and histones H3-H4. Biochemical analyses establish that POLE3-POLE4 is a histone chaperone that promotes tetrasome formation and DNA supercoiling in vitro. In cells, POLE3-POLE4 binds both newly synthesized and parental histones, and its depletion hinders helicase unwinding and chromatin PCNA unloading and compromises coordinated parental histone retention and new histone deposition. Collectively, our study reveals that POLE3-POLE4 possesses intrinsic H3-H4 chaperone activity, which facilitates faithful nucleosome dynamics at the replication fork.
Assuntos
DNA Polimerase III/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Epigênese Genética/genética , Histonas/biossíntese , Nucleoproteínas/genética , Cromatina/genética , DNA Polimerase II/química , DNA Polimerase II/genética , DNA Polimerase III/química , Proteínas de Ligação a DNA/química , Chaperonas de Histonas/química , Chaperonas de Histonas/genética , Histonas/genética , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Nucleoproteínas/química , Nucleossomos/química , Nucleossomos/genética , Proteínas de Ligação a Poli-ADP-Ribose/química , Proteínas de Ligação a Poli-ADP-Ribose/genética , Antígeno Nuclear de Célula em Proliferação/genética , Ligação ProteicaRESUMO
Protein post-translation modification plays an important role in regulating DNA repair; however, the role of arginine methylation in this process is poorly understood. Here we identify the arginine methyltransferase PRMT5 as a key regulator of homologous recombination (HR)-mediated double-strand break (DSB) repair, which is mediated through its ability to methylate RUVBL1, a cofactor of the TIP60 complex. We show that PRMT5 targets RUVBL1 for methylation at position R205, which facilitates TIP60-dependent mobilization of 53BP1 from DNA breaks, promoting HR. Mechanistically, we demonstrate that PRMT5-directed methylation of RUVBL1 is critically required for the acetyltransferase activity of TIP60, promoting histone H4K16 acetylation, which facilities 53BP1 displacement from DSBs. Interestingly, RUVBL1 methylation did not affect the ability of TIP60 to facilitate ATM activation. Taken together, our findings reveal the importance of PRMT5-mediated arginine methylation during DSB repair pathway choice through its ability to regulate acetylation-dependent control of 53BP1 localization.
Assuntos
Proteínas de Transporte/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Histona Acetiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/metabolismo , Reparo de DNA por Recombinação , ATPases Associadas a Diversas Atividades Celulares , Acetilação , Animais , Arginina , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte/genética , DNA Helicases/genética , Instabilidade Genômica , Células HEK293 , Células HeLa , Histona Acetiltransferases/genética , Histonas/metabolismo , Humanos , Lisina Acetiltransferase 5 , Metilação , Camundongos , Camundongos Transgênicos , Proteína-Arginina N-Metiltransferases/genética , Interferência de RNA , Fatores de Tempo , Transfecção , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
Human ALC1 is an oncogene-encoded chromatin-remodeling enzyme required for DNA repair that possesses a poly(ADP-ribose) (PAR)-binding macro domain. Its engagement with PARylated PARP1 activates ALC1 at sites of DNA damage, but the underlying mechanism remains unclear. Here, we establish a dual role for the macro domain in autoinhibition of ALC1 ATPase activity and coupling to nucleosome mobilization. In the absence of DNA damage, an inactive conformation of the ATPase is maintained by juxtaposition of the macro domain against predominantly the C-terminal ATPase lobe through conserved electrostatic interactions. Mutations within this interface displace the macro domain, constitutively activate the ALC1 ATPase independent of PARylated PARP1, and alter the dynamics of ALC1 recruitment at DNA damage sites. Upon DNA damage, binding of PARylated PARP1 by the macro domain induces a conformational change that relieves autoinhibitory interactions with the ATPase motor, which selectively activates ALC1 remodeling upon recruitment to sites of DNA damage.
Assuntos
Montagem e Desmontagem da Cromatina , Dano ao DNA , DNA Helicases/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Nucleossomos/enzimologia , Domínio Catalítico , Linhagem Celular Tumoral , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Humanos , Microscopia Eletrônica , Simulação de Dinâmica Molecular , Mutação , Nucleossomos/química , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli ADP Ribosilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Espalhamento a Baixo Ângulo , Eletricidade Estática , Relação Estrutura-Atividade , Fatores de Tempo , Difração de Raios XRESUMO
Mutations in the E3 ubiquitin ligase parkin (PARK2, also known as PRKN) and the protein kinase PINK1 (also known as PARK6) are linked to autosomal-recessive juvenile parkinsonism (AR-JP)1,2; at the cellular level, these mutations cause defects in mitophagy, the process that organizes the destruction of damaged mitochondria3,4. Parkin is autoinhibited, and requires activation by PINK1, which phosphorylates Ser65 in ubiquitin and in the parkin ubiquitin-like (Ubl) domain. Parkin binds phospho-ubiquitin, which enables efficient parkin phosphorylation; however, the enzyme remains autoinhibited with an inaccessible active site5,6. It is unclear how phosphorylation of parkin activates the molecule. Here we follow the activation of full-length human parkin by hydrogen-deuterium exchange mass spectrometry, and reveal large-scale domain rearrangement in the activation process, during which the phospho-Ubl rebinds to the parkin core and releases the catalytic RING2 domain. A 1.8 Å crystal structure of phosphorylated human parkin reveals the binding site of the phospho-Ubl on the unique parkin domain (UPD), involving a phosphate-binding pocket lined by AR-JP mutations. Notably, a conserved linker region between Ubl and the UPD acts as an activating element (ACT) that contributes to RING2 release by mimicking RING2 interactions on the UPD, explaining further AR-JP mutations. Our data show how autoinhibition in parkin is resolved, and suggest a mechanism for how parkin ubiquitinates its substrates via an untethered RING2 domain. These findings open new avenues for the design of parkin activators for clinical use.
Assuntos
Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sítios de Ligação , Medição da Troca de Deutério , Ativação Enzimática , Humanos , Espectrometria de Massas , Modelos Moleculares , Fosforilação , Domínios Proteicos , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/química , UbiquitinaçãoRESUMO
Covalent DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with essential chromatin transactions, such as replication and transcription. Little was known about DPC-specific repair mechanisms until the recent identification of a DPC-processing protease in yeast. The existence of a DPC protease in higher eukaryotes is inferred from data in Xenopus laevis egg extracts, but its identity remains elusive. Here we identify the metalloprotease SPRTN as the DPC protease acting in metazoans. Loss of SPRTN results in failure to repair DPCs and hypersensitivity to DPC-inducing agents. SPRTN accomplishes DPC processing through a unique DNA-induced protease activity, which is controlled by several sophisticated regulatory mechanisms. Cellular, biochemical, and structural studies define a DNA switch triggering its protease activity, a ubiquitin switch controlling SPRTN chromatin accessibility, and regulatory autocatalytic cleavage. Our data also provide a molecular explanation on how SPRTN deficiency causes the premature aging and cancer predisposition disorder Ruijs-Aalfs syndrome.
Assuntos
Proteínas de Caenorhabditis elegans/química , Reparo do DNA , Proteínas de Ligação a DNA/química , DNA/química , Proteínas de Schizosaccharomyces pombe/química , Proteína de Xeroderma Pigmentoso Grupo A/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/efeitos da radiação , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Linhagem Celular , Cisplatino/química , Reagentes de Ligações Cruzadas/química , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/efeitos da radiação , Formaldeído/química , Células HeLa , Humanos , Cinética , Camundongos , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Raios Ultravioleta , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismoRESUMO
Autosomal-recessive juvenile Parkinsonism (AR-JP) is caused by mutations in a number of PARK genes, in particular the genes encoding the E3 ubiquitin ligase Parkin (PARK2, also known as PRKN) and its upstream protein kinase PINK1 (also known as PARK6). PINK1 phosphorylates both ubiquitin and the ubiquitin-like domain of Parkin on structurally protected Ser65 residues, triggering mitophagy. Here we report a crystal structure of a nanobody-stabilized complex containing Pediculus humanus corporis (Ph)PINK1 bound to ubiquitin in the 'C-terminally retracted' (Ub-CR) conformation. The structure reveals many peculiarities of PINK1, including the architecture of the C-terminal region, and reveals how the N lobe of PINK1 binds ubiquitin via a unique insertion. The flexible Ser65 loop in the Ub-CR conformation contacts the activation segment, facilitating placement of Ser65 in a phosphate-accepting position. The structure also explains how autophosphorylation in the N lobe stabilizes structurally and functionally important insertions, and reveals the molecular basis of AR-JP-causing mutations, some of which disrupt ubiquitin binding.
Assuntos
Pediculus/enzimologia , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Animais , Sítios de Ligação , Cristalografia por Raios X , Mitofagia , Modelos Moleculares , Mutação , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/imunologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologiaRESUMO
The post-translational modification of proteins with polyubiquitin regulates virtually all aspects of cell biology. Eight distinct chain linkage types co-exist in polyubiquitin and are independently regulated in cells. This 'ubiquitin code' determines the fate of the modified protein. Deubiquitinating enzymes of the ovarian tumour (OTU) family regulate cellular signalling by targeting distinct linkage types within polyubiquitin, and understanding their mechanisms of linkage specificity gives fundamental insights into the ubiquitin system. Here we reveal how the deubiquitinase Cezanne (also known as OTUD7B) specifically targets Lys11-linked polyubiquitin. Crystal structures of Cezanne alone and in complex with monoubiquitin and Lys11-linked diubiquitin, in combination with hydrogen-deuterium exchange mass spectrometry, enable us to reconstruct the enzymatic cycle in great detail. An intricate mechanism of ubiquitin-assisted conformational changes activates the enzyme, and while all chain types interact with the enzymatic S1 site, only Lys11-linked chains can bind productively across the active site and stimulate catalytic turnover. Our work highlights the plasticity of deubiquitinases and indicates that new conformational states can occur when a true substrate, such as diubiquitin, is bound at the active site.
Assuntos
Enzimas Desubiquitinantes/metabolismo , Endopeptidases/metabolismo , Lisina/metabolismo , Poliubiquitina/metabolismo , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/genética , Medição da Troca de Deutério , Endopeptidases/química , Endopeptidases/genética , Ativação Enzimática , Humanos , Espectrometria de Massas , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Especificidade por Substrato , Ubiquitinação , Ubiquitinas/metabolismoRESUMO
Many natural metalloenzymes assemble from proteins and biosynthesised complexes, generating potent catalysts by changing metal coordination. Here we adopt the same strategy to generate artificial metalloenzymes (ArMs) using ligand exchange to unmask catalytic activity. By systematically testing RuII (η6 -arene)(bipyridine) complexes designed to facilitate the displacement of functionalised bipyridines, we develop a fast and robust procedure for generating new enzymes via ligand exchange in a protein that has not evolved to bind such a complex. The resulting metal cofactors form peptidic coordination bonds but also retain a non-biological ligand. Tandem mass spectrometry and 19 Fâ NMR spectroscopy were used to characterise the organometallic cofactors and identify the protein-derived ligands. By introduction of ruthenium cofactors into a 4-helical bundle, transfer hydrogenation catalysts were generated that displayed a 35-fold rate increase when compared to the respective small molecule reaction in solution.
Assuntos
Metaloproteínas/metabolismo , Compostos Organometálicos/química , Rutênio/química , Catálise , Flúor , Hidrogenação , Ligantes , Espectroscopia de Ressonância Magnética , Metaloproteínas/química , Estrutura Molecular , Compostos Organometálicos/metabolismo , Rutênio/metabolismoRESUMO
The protein ProQ has recently been identified as a global small noncoding RNA-binding protein in Salmonella, and a similar role is anticipated for its numerous homologs in divergent bacterial species. We report the solution structure of Escherichia coli ProQ, revealing an N-terminal FinO-like domain, a C-terminal domain that unexpectedly has a Tudor domain fold commonly found in eukaryotes, and an elongated bridging intradomain linker that is flexible but nonetheless incompressible. Structure-based sequence analysis suggests that the Tudor domain was acquired through horizontal gene transfer and gene fusion to the ancestral FinO-like domain. Through a combination of biochemical and biophysical approaches, we have mapped putative RNA-binding surfaces on all three domains of ProQ and modeled the protein's conformation in the apo and RNA-bound forms. Taken together, these data suggest how the FinO, Tudor, and linker domains of ProQ cooperate to recognize complex RNA structures and serve to promote RNA-mediated regulation.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Ligação a RNA/química , Regiões 3' não Traduzidas , Sítios de Ligação , Proteínas de Escherichia coli/metabolismo , Fator Proteico 1 do Hospedeiro/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Proteínas de Ligação a RNA/metabolismoRESUMO
Human Timeless is involved in replication fork stabilization, S-phase checkpoint activation and establishment of sister chromatid cohesion. In the cell, Timeless forms a constitutive heterodimeric complex with Tipin. Here we present the 1.85 Å crystal structure of a large N-terminal segment of human Timeless, spanning amino acids 1-463, and we show that this region of human Timeless harbours a partial binding site for Tipin. Furthermore, we identify minimal regions of the two proteins that are required for the formation of a stable Timeless-Tipin complex and provide evidence that the Timeless-Tipin interaction is based on a composite binding interface comprising different domains of Timeless.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Fenômenos Biofísicos , Reagentes de Ligações Cruzadas/metabolismo , Cristalografia por Raios X , Proteínas de Ligação a DNA , Humanos , Espectrometria de Massas , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Homologia Estrutural de ProteínaRESUMO
Until recently, one of the major limitations of hydrogen/deuterium exchange mass spectrometry (HDX-MS) was the peptide-level resolution afforded by proteolytic digestion. This limitation can be selectively overcome through the use of electron-transfer dissociation to fragment peptides in a manner that allows the retention of the deuterium signal to produce hydrogen/deuterium exchange tandem mass spectrometry (HDX-MS/MS). Here, we describe the application of HDX-MS/MS to structurally screen inhibitors of the oncogene phosphoinositide 3-kinase catalytic p110α subunit. HDX-MS/MS analysis is able to discern a conserved mechanism of inhibition common to a range of inhibitors. Owing to the relatively minor amounts of protein required, this technique may be utilised in pharmaceutical development for screening potential therapeutics.
Assuntos
Antineoplásicos/metabolismo , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Inibidores Enzimáticos/metabolismo , Modelos Moleculares , Fragmentos de Peptídeos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Classe I de Fosfatidilinositol 3-Quinases , Classe Ia de Fosfatidilinositol 3-Quinase/química , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Medição da Troca de Deutério , Avaliação Pré-Clínica de Medicamentos/métodos , Transporte de Elétrons , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Indazóis/química , Indazóis/metabolismo , Indazóis/farmacologia , Peso Molecular , Oligonucleotídeos/antagonistas & inibidores , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Conformação Proteica , Purinas/química , Purinas/metabolismo , Purinas/farmacologia , Piridazinas , Quinazolinonas/química , Quinazolinonas/metabolismo , Quinazolinonas/farmacologia , Quinolinas/química , Quinolinas/metabolismo , Quinolinas/farmacologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , Processamento de Sinais Assistido por Computador , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacologia , Espectrometria de Massas em Tandem , Triazinas/química , Triazinas/metabolismo , Triazinas/farmacologiaRESUMO
The Fanconi anemia (FA) pathway is implicated in DNA repair and cancer predisposition. Central to this pathway is the FA core complex, which is targeted to chromatin by FANCM and FAAP24 following replication stress. Here we show that FANCM and FAAP24 interact with the checkpoint protein HCLK2 independently of the FA core complex. In addition to defects in FA pathway activation, downregulation of FANCM or FAAP24 also compromises ATR/Chk1-mediated checkpoint signaling, leading to defective Chk1, p53, and FANCE phosphorylation; 53BP1 focus formation; and Cdc25A degradation. As a result, FANCM and FAAP24 deficiency results in increased endogenous DNA damage and a failure to efficiently invoke cell-cycle checkpoint responses. Moreover, we find that the DNA translocase activity of FANCM, which is dispensable for FA pathway activation, is required for its role in ATR/Chk1 signaling. Our data suggest that DNA damage recognition and remodeling activities of FANCM and FAAP24 cooperate with ATR/Chk1 to promote efficient activation of DNA damage checkpoints.
Assuntos
Dano ao DNA , DNA Helicases/genética , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/genética , Anemia de Fanconi/genética , Linhagem Celular , DNA Helicases/deficiência , DNA Helicases/isolamento & purificação , DNA Helicases/metabolismo , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi , Genoma , Células HeLa , Humanos , Rim , Mitose , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/isolamento & purificação , Proteínas Tirosina Quinases/metabolismo , Fase S , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Photoaffinity labelling is a promising method for studying protein-ligand interactions. However, obtaining a specific, efficient crosslinker can require significant optimisation. We report a modified mRNA display strategy, photocrosslinking-RaPID (XL-RaPID), and exploit its ability to accelerate the discovery of cyclic peptides that photocrosslink to a target of interest. As a proof of concept, we generated a benzophenone-containing library and applied XL-RaPID screening against a model target, the second bromodomain of BRD3. This crosslinking screening gave two optimal candidates that selectively labelled the target protein in cell lysate. Overall, this work introduces direct photocrosslinking screening as a versatile technique for identifying covalent peptide ligands from mRNA display libraries incorporating reactive warheads.
RESUMO
TRIM proteins are the largest family of E3 ligases in mammals. They include the intracellular antibody receptor TRIM21, which is responsible for mediating targeted protein degradation during Trim-Away. Despite their importance, the ubiquitination mechanism of TRIM ligases has remained elusive. Here we show that while Trim-Away activation results in ubiquitination of both ligase and substrate, ligase ubiquitination is not required for substrate degradation. N-terminal TRIM21 RING ubiquitination by the E2 Ube2W can be inhibited by N-terminal acetylation, but this doesn't prevent substrate ubiquitination nor degradation. Instead, uncoupling ligase and substrate degradation prevents ligase recycling and extends functional persistence in cells. Further, Trim-Away degrades substrates irrespective of whether they contain lysines or are N-terminally acetylated, which may explain the ability of TRIM21 to counteract fast-evolving pathogens and degrade diverse substrates.