RESUMO
BACKGROUND: The sarcoplasmic reticulum (SR) Ca2+-ATPase 2 (SERCA2) mediates Ca2+ reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca2+ release from SR and triggers contraction. Ca2+/CaMKII (CaM [calmodulin]-dependent protein kinase II) regulates activities of SERCA2 through phosphorylation of PLN (phospholamban) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local Ca2+ signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR. METHODS: A role for AKAP18δ (A-kinase anchoring protein 18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adenoassociated virus injection, structural modeling, surface plasmon resonance, and alpha screen technology. RESULTS: Our results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via 2 distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to the natural CaMKII inhibitor peptide and the Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent Ca2+ threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster Ca2+ reuptake by SERCA2 and Ca2+ release through RYR, AKAP18δ-N had opposite effects. We propose a model where the 2 unique AKAP18δ regions fine-tune Ca2+-frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR. CONCLUSIONS: AKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge, this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP (A-kinase anchoring protein) reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Animais , Sítios de Ligação , Sinalização do Cálcio , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Células Cultivadas , Células HEK293 , Humanos , Miócitos Cardíacos/metabolismo , Ligação Proteica , Ratos , Ratos WistarRESUMO
RNA polymerase I transcribes ribosomal DNA to produce precursor 47S rRNA. Post-transcriptional processing of this rRNA generates mature 28S, 18S and 5.8S rRNAs, which form the ribosomes, together with 5S rRNA, assembly factors and ribosomal proteins. We previously reported a homozygous variant in the catalytic subunit of RNA polymerase I, POLR1A, in two brothers with leukodystrophy and progressive course. However, the disease mechanism remained unknown. In this report, we describe another missense variant POLR1A NM_015425.3:c.1925C>A; p.(Thr642Asn) in homozygosity in two unrelated patients. Patient 1 was a 16-year-old male and Patient 2 was a 2-year-old female. Both patients manifested neurological deficits, with brain MRIs showing hypomyelinating leukodystrophy and cerebellar atrophy; and in Patient 1 additionally with hypointensity of globi pallidi and small volume of the basal ganglia. Patient 1 had progressive disease course, leading to death at the age of 16.5 years. Extensive in vitro experiments in fibroblasts from Patient 1 documented that the mutated POLR1A led to aberrant rRNA processing and degradation, and abnormal nucleolar homeostasis. Proteomics data analyses and further in vitro experiments documented abnormal protein homeostasis, and endoplasmic reticulum stress responses. We confirm that POLR1A biallelic variants cause neurodegenerative disease, expand the knowledge of the clinical phenotype of the disorder, and provide evidence for possible pathological mechanisms leading to POLR1A-related leukodystrophy.
Assuntos
Doenças Neurodegenerativas , RNA Polimerase I , Masculino , Feminino , Humanos , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , Doenças Neurodegenerativas/genética , Proteostase , RNA Ribossômico/metabolismo , Ribossomos , Processamento Pós-Transcricional do RNARESUMO
In the fight against antimicrobial resistance, the bacterial DNA sliding clamp, ß-clamp, is a promising drug target for inhibition of DNA replication and translesion synthesis. The ß-clamp and its eukaryotic homolog, PCNA, share a C-terminal hydrophobic pocket where all the DNA polymerases bind. Here we report that cell penetrating peptides containing the PCNA-interacting motif APIM (APIM-peptides) inhibit bacterial growth at low concentrations in vitro, and in vivo in a bacterial skin infection model in mice. Surface plasmon resonance analysis and computer modeling suggest that APIM bind to the hydrophobic pocket on the ß-clamp, and accordingly, we find that APIM-peptides inhibit bacterial DNA replication. Interestingly, at sub-lethal concentrations, APIM-peptides have anti-mutagenic activities, and this activity is increased after SOS induction. Our results show that although the sequence homology between the ß-clamp and PCNA are modest, the presence of similar polymerase binding pockets in the DNA clamps allows for binding of the eukaryotic binding motif APIM to the bacterial ß-clamp. Importantly, because APIM-peptides display both anti-mutagenic and growth inhibitory properties, they may have clinical potential both in combination with other antibiotics and as single agents.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , DNA Polimerase III/antagonistas & inibidores , Peptídeos/química , Peptídeos/farmacologia , Animais , Antibacterianos/metabolismo , Antibacterianos/uso terapêutico , DNA Polimerase III/química , Replicação do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA , Feminino , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Camundongos Endogâmicos BALB C , Mutagênese/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/química , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores da Síntese de Ácido Nucleico/uso terapêutico , Peptídeos/metabolismo , Peptídeos/uso terapêutico , Antígeno Nuclear de Célula em Proliferação/metabolismo , Domínios e Motivos de Interação entre Proteínas , Infecções Cutâneas Estafilocócicas/tratamento farmacológico , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/crescimento & desenvolvimentoRESUMO
Single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1) is a base excision repair enzyme that removes uracil and oxidised pyrimidines from DNA. We show that SMUG1 interacts with the pseudouridine synthase Dyskerin (DKC1) and colocalizes with DKC1 in nucleoli and Cajal bodies. As DKC1 functions in RNA processing, we tested whether SMUG1 excised modified bases in RNA and demonstrated that SMUG1 has activity on single-stranded RNA containing 5-hydroxymethyldeoxyuridine, but not pseudouridine, the nucleoside resulting from isomerization of uridine by DKC1. Moreover, SMUG1 associates with the 47S rRNA precursor processed by DKC1, and depletion of SMUG1 leads to a reduction in the levels of mature rRNA accompanied by an increase in polyadenylated rRNA. Depletion of SMUG1, and, in particular, the combined loss of SMUG1 and DKC1, leads to accumulation of 5-hydroxymethyluridine in rRNA. In conclusion, SMUG1 is a DKC1 interaction partner that contributes to rRNA quality control, partly by regulating 5-hydroxymethyluridine levels.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Processamento Pós-Transcricional do RNA , Uracila-DNA Glicosidase/metabolismo , Proteínas de Ciclo Celular/genética , Nucléolo Celular/metabolismo , Corpos Enovelados/metabolismo , Biblioteca Gênica , Células HeLa , Humanos , Proteínas Nucleares/genética , Poliadenilação , Ligação Proteica , Mapeamento de Interação de Proteínas , Transporte Proteico , RNA Ribossômico 18S/metabolismo , RNA Ribossômico 28S/metabolismo , RNA Interferente Pequeno/genética , Uracila-DNA Glicosidase/genética , Uridina/análogos & derivados , Uridina/metabolismoRESUMO
Bacterial lytic polysaccharide monooxygenases (LPMO10s) use redox chemistry to cleave glycosidic bonds in the two foremost recalcitrant polysaccharides found in nature, namely cellulose and chitin. Analysis of correlated mutations revealed that the substrate-binding and copper-containing surface of LPMO10s composes a network of co-evolved residues and interactions, whose roles in LPMO functionality are unclear. Here, we mutated a subset of these correlated residues in a newly characterized C1/C4-oxidizing LPMO10 from Micromonospora aurantiaca (MaLPMO10B) to the corresponding residues in strictly C1-oxidizing LPMO10s. We found that surface properties near the catalytic copper, i.e. side chains likely to be involved in substrate positioning, are major determinants of the C1:C4 ratio. Several MaLPMO10B mutants almost completely lost C4-oxidizing activity while maintaining C1-oxidizing activity. These mutants also lost chitin-oxidizing activity, which is typically observed for C1/C4-oxidizing, but not for C1-oxidizing, cellulose-active LPMO10s. Selective loss in C1-oxidizing activity was not observed. Additional mutational experiments disclosed that neither truncation of the MaLPMO10B family 2 carbohydrate-binding module nor mutations altering access to the solvent-exposed axial copper coordination site significantly change the C1:C4 ratio. Importantly, several of the mutations that altered interactions with the substrate exhibited reduced stability. This effect could be explained by productive substrate binding that protects LPMOs from oxidative self-inactivation. We discuss these stability issues in view of recent findings on LPMO catalysis, such as the involvement of H2O2 Our results show that residues on the substrate-binding surface of LPMOs have co-evolved to optimize several of the interconnected properties: substrate binding and specificity, oxidative regioselectivity, catalytic efficiency, and stability.
Assuntos
Proteínas de Bactérias/química , Peróxido de Hidrogênio/química , Micromonospora/enzimologia , Oxigenases de Função Mista/química , Catálise , Oxirredução , Domínios Proteicos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Oncogenic mutations of the Wnt (wingless)/ß-catenin pathway are frequently observed in major cancer types. Thus far, however, no therapeutic agent targeting Wnt/ß-catenin signaling is available for clinical use. Here we demonstrate that axitinib, a clinically approved drug, strikingly blocks Wnt/ß-catenin signaling in cancer cells, zebrafish, and Apc(min/+) mice. Notably, axitinib dramatically induces Wnt asymmetry and nonrandom DNA segregation in cancer cells by promoting nuclear ß-catenin degradation independent of the GSK3ß (glycogen synthase kinase3ß)/APC (adenomatous polyposis coli) complex. Using a DARTS (drug affinity-responsive target stability) assay coupled to 2D-DIGE (2D difference in gel electrophoresis) and mass spectrometry, we have identified the E3 ubiquitin ligase SHPRH (SNF2, histone-linker, PHD and RING finger domain-containing helicase) as the direct target of axitinib in blocking Wnt/ß-catenin signaling. Treatment with axitinib stabilizes SHPRH and thereby increases the ubiquitination and degradation of ß-catenin. Our findings suggest a previously unreported mechanism of nuclear ß-catenin regulation and indicate that axitinib, a clinically approved drug, would provide therapeutic benefits for cancer patients with aberrant nuclear ß-catenin activation.
Assuntos
Divisão Celular/efeitos dos fármacos , Imidazóis/farmacologia , Indazóis/farmacologia , Neoplasias/patologia , Inibidores de Proteínas Quinases/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/fisiologia , Animais , Axitinibe , DNA Helicases/fisiologia , Glicogênio Sintase Quinase 3 beta/fisiologia , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regeneração/efeitos dos fármacos , Ubiquitina-Proteína Ligases/fisiologia , Peixe-ZebraRESUMO
Understanding features that determine transglycosylation (TG) activity in glycoside hydrolases is important because it would allow the construction of enzymes that can catalyze controlled synthesis of oligosaccharides. To increase TG activity in two family 18 chitinases, chitinase D from Serratia proteamaculans ( SpChiD) and chitinase A from Serratia marcescens ( SmChiA), we have mutated residues important for stabilizing the reaction intermediate and substrate binding in both donor and acceptor sites. To help mutant design, the crystal structure of the inactive SpChiD-E153Q mutant in complex with chitobiose was determined. We identified three mutations with a beneficial effect on TG activity: Y28A (affecting the -1 subsite and the intermediate), Y222A (affecting the intermediate), and Y226W (affecting the +2 subsite). Furthermore, exchange of D151, the middle residue in the catalytically important DXDXE motif, to asparagine reduced hydrolytic activity ≤99% with a concomitant increase in apparent TG activity. The combination of mutations yielded even higher degrees of TG activity. Reactions with the best mutant, SpChiD-D151N/Y226W/Y222A, led to rapid accumulation of high levels of TG products that remained stable over time. Importantly, the introduction of analogous mutations at the same positions in SmChiA (Y163A equal to Y28A and Y390F similar to Y222A) had similar effects on TG efficiency. Thus, the combination of the decreasing hydrolytic power, subsite affinity, and stability of intermediate states provides a powerful, general strategy for creating hypertransglycosylating mutants of retaining glycoside hydrolases.
Assuntos
Quitinases/química , Quitinases/metabolismo , Serratia marcescens/enzimologia , Sequência de Aminoácidos , Quitinases/genética , Cristalografia por Raios X , Dissacarídeos/metabolismo , Glicosilação , Hidrólise , Modelos Moleculares , Mutação , Alinhamento de Sequência , Serratia/química , Serratia/enzimologia , Serratia/metabolismo , Infecções por Serratia/microbiologia , Serratia marcescens/química , Serratia marcescens/genética , Serratia marcescens/metabolismoRESUMO
Ab-coated viruses can be detected in the cytosol by the FcR tripartite motif-containing 21 (TRIM21), which rapidly recruits the proteasomal machinery and triggers induction of immune signaling. As such, TRIM21 plays a key role in intracellular protection by targeting invading viruses for destruction and alerting the immune system. A hallmark of immunity is elicitation of a balanced response that is proportionate to the threat, to avoid unnecessary inflammation. In this article, we show how Ab affinity modulates TRIM21 immune function. We constructed a humanized monoclonal IgG1 against human adenovirus type 5 (AdV5) and a panel of Fc-engineered variants with a wide range of affinities for TRIM21. We found that IgG1-coated viral particles were neutralized via TRIM21, even when affinity was reduced by as much as 100-fold. In contrast, induction of NF-κB signaling was more sensitive to reduced affinity between TRIM21 and the Ab variants. Thus, TRIM21 mediates neutralization under suboptimal conditions, whereas induction of immune signaling is balanced according to the functional affinity for the incoming immune stimuli. Our findings have implications for engineering of antiviral IgG therapeutics with tailored effector functions.
Assuntos
Adenovírus Humanos/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Neutralizantes/imunologia , Afinidade de Anticorpos/imunologia , Imunoglobulina G/imunologia , Ribonucleoproteínas/imunologia , Animais , Linhagem Celular , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/imunologia , Testes de Neutralização , Ribonucleoproteínas/genética , Transdução de Sinais/imunologia , Ressonância de Plasmônio de SuperfícieRESUMO
Unstable hemoglobin (Hb) variants are the result of sequence variants in the globin genes causing precipitation of Hb molecules in red blood cells (RBCs). Intracellular inclusions derived from the unstable Hb reduce the life-span of the red cells and may cause hemolytic anemia. Here we describe a patient with a history of hemolytic anemia and low oxygen saturation. She was found to be carrier of a novel unstable Hb variant, Hb Oslo [ß42(CD1)PheâIle (TTT>ATT), HBB: c.127T>A] located in the heme pocket of the ß-globin chain. Three-dimensional modeling suggested that isoleucine at position 42 creates weaker interactions with distal histidine and with the heme itself, which may lead to altered stability and decreased oxygen affinity. At steady state, the patient was in good clinical condition with a Hb concentration of 8.0-9.0 g/dL. During virus infections, the Hb concentration fell and on six occasions during 4 years, the patient needed a blood transfusion.
Assuntos
Anemia Hemolítica/genética , Hemoglobinopatias/genética , Hemoglobinas Anormais/genética , Mutação de Sentido Incorreto , Transfusão de Sangue , Precipitação Química , Feminino , Humanos , Noruega , Viroses/etiologia , Viroses/terapia , Globinas beta/genéticaRESUMO
Endonuclease V (EndoV) is an enzyme with specificity for inosines in nucleic acids. Whereas the bacterial homologs are active on both DNA and RNA, the mammalian variants only cleave RNA, at least when assayed with recombinant proteins. Here we show that ectopically expressed, as well as endogenously expressed human (h)EndoV, share the same enzymatic properties as the recombinant protein and cleaves RNA with inosine but not DNA. In search for proteins interacting with hEndoV, polyadenylate-binding protein C1 (PABPC1) was identified. The association between PABPC1 and hEndoV is RNA dependent and furthermore, PABPC1 stimulates hEndoV activity and affinity for inosine-containing RNA. Upon cellular stress, PABPC1 relocates to cytoplasmic stress granules that are multimolecular aggregates of stalled translation initiation complexes formed to aid cell recovery. Arsenite, as well as other agents, triggered relocalization also of hEndoV to cytoplasmic stress granules. As inosines in RNA are highly abundant, hEndoV activity is likely regulated in cells to avoid aberrant cleavage of inosine-containing transcripts. Indeed, we find that hEndoV cleavage is inhibited by normal intracellular ATP concentrations. The ATP stores inside a cell do not overlay stress granules and we suggest that hEndoV is redistributed to stress granules as a strategy to create a local environment low in ATP to permit hEndoV activity.
Assuntos
Trifosfato de Adenosina/metabolismo , Grânulos Citoplasmáticos/enzimologia , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , RNA/metabolismo , Trifosfato de Adenosina/genética , Arsenitos/farmacologia , Grânulos Citoplasmáticos/genética , Desoxirribonuclease (Dímero de Pirimidina)/genética , Células HEK293 , Células HeLa , Humanos , Proteína I de Ligação a Poli(A)/genética , Proteína I de Ligação a Poli(A)/metabolismo , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , RNA/genéticaRESUMO
Cellvibrio japonicusis a Gram-negative soil bacterium that is primarily known for its ability to degrade plant cell wall polysaccharides through utilization of an extensive repertoire of carbohydrate-active enzymes. Several putative chitin-degrading enzymes are also found among these carbohydrate-active enzymes, such as chitinases, chitobiases, and lytic polysaccharide monooxygenases (LPMOs). In this study, we have characterized the chitin-active LPMO,CjLPMO10A, a tri-modular enzyme containing a catalytic family AA10 LPMO module, a family 5 chitin-binding module, and a C-terminal unclassified module of unknown function. Characterization of the latter module revealed tight and specific binding to chitin, thereby unraveling a new family of chitin-binding modules (classified as CBM73). X-ray crystallographic elucidation of theCjLPMO10A catalytic module revealed that the active site of the enzyme combines structural features previously only observed in either cellulose or chitin-active LPMO10s. Analysis of the copper-binding site by EPR showed a signal signature more similar to those observed for cellulose-cleaving LPMOs. The full-length LPMO shows no activity toward cellulose but is able to bind and cleave both α- and ß-chitin. Removal of the chitin-binding modules reduced LPMO activity toward α-chitin compared with the full-length enzyme. Interestingly, the full-length enzyme and the individual catalytic LPMO module boosted the activity of an endochitinase equally well, also yielding similar amounts of oxidized products. Finally, gene deletion studies show thatCjLPMO10A is needed byC. japonicusto obtain efficient growth on both purified chitin and crab shell particles.
Assuntos
Cellvibrio/enzimologia , Quitina/química , Oxigenases de Função Mista/química , Quitina/metabolismo , Cristalografia por Raios X , Oxigenases de Função Mista/metabolismo , Estrutura Terciária de ProteínaRESUMO
The sodium (Na(+))-calcium (Ca(2+)) exchanger 1 (NCX1) is an important regulator of intracellular Ca(2+) homeostasis. Serine 68-phosphorylated phospholemman (pSer-68-PLM) inhibits NCX1 activity. In the context of Na(+)/K(+)-ATPase (NKA) regulation, pSer-68-PLM is dephosphorylated by protein phosphatase 1 (PP1). PP1 also associates with NCX1; however, the molecular basis of this association is unknown. In this study, we aimed to analyze the mechanisms of PP1 targeting to the NCX1-pSer-68-PLM complex and hypothesized that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer-68-PLM dephosphorylation. Using a variety of molecular techniques, we show that PP1 catalytic subunit (PP1c) co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes, left ventricle lysates, and HEK293 cells. Bioinformatic analysis, immunoprecipitations, mutagenesis, pulldown experiments, and peptide arrays constrained PP1c anchoring to the K(I/V)FF motif in the first Ca(2+) binding domain (CBD) 1 in NCX1. This binding site is also partially in agreement with the extended PP1-binding motif K(V/I)FF-X5-8Φ1Φ2-X8-9-R. The cytosolic loop of NCX1, containing the K(I/V)FF motif, had no effect on PP1 activity in an in vitro assay. Dephosphorylation of pSer-68-PLM in HEK293 cells was not observed when NCX1 was absent, when the K(I/V)FF motif was mutated, or when the PLM- and PP1c-binding sites were separated (mimicking calpain cleavage of NCX1). Co-expression of PLM and NCX1 inhibited NCX1 current (both modes). Moreover, co-expression of PLM with NCX1(F407P) (mutated K(I/V)FF motif) resulted in the current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation.
Assuntos
Modelos Animais de Doenças , Insuficiência Cardíaca/metabolismo , Proteínas de Membrana/metabolismo , Miócitos Cardíacos/metabolismo , Fosfoproteínas/metabolismo , Proteína Fosfatase 1/metabolismo , Processamento de Proteína Pós-Traducional , Trocador de Sódio e Cálcio/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Biologia Computacional , Células HEK293 , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/patologia , Humanos , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Proteína Fosfatase 1/química , Proteína Fosfatase 1/genética , Ratos Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/genética , Especificidade por SubstratoRESUMO
The functions of several SOS regulated genes in Escherichia coli are still unknown, including dinQ. In this work we characterize dinQ and two small RNAs, agrA and agrB, with antisense complementarity to dinQ. Northern analysis revealed five dinQ transcripts, but only one transcript (+44) is actively translated. The +44 dinQ transcript translates into a toxic single transmembrane peptide localized in the inner membrane. AgrB regulates dinQ RNA by RNA interference to counteract DinQ toxicity. Thus the dinQ-agr locus shows the classical features of a type I TA system and has many similarities to the tisB-istR locus. DinQ overexpression depolarizes the cell membrane and decreases the intracellular ATP concentration, demonstrating that DinQ can modulate membrane-dependent processes. Augmented DinQ strongly inhibits marker transfer by Hfr conjugation, indicating a role in recombination. Furthermore, DinQ affects transformation of nucleoid morphology in response to UV damage. We hypothesize that DinQ is a transmembrane peptide that modulates membrane-dependent activities such as nucleoid compaction and recombination.
Assuntos
Membrana Celular , Proteínas de Escherichia coli/genética , Escherichia coli , Proteínas de Membrana/genética , RNA Bacteriano , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/efeitos da radiação , Citoplasma , Dano ao DNA/efeitos da radiação , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Peptídeos/genética , Peptídeos/metabolismo , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Recombinação Genética/genética , Resposta SOS em Genética/efeitos da radiação , Transativadores/genética , Transativadores/metabolismo , Raios UltravioletaRESUMO
Albumin is an abundant blood protein that acts as a transporter of a plethora of small molecules like fatty acids, hormones, toxins, and drugs. In addition, it has an unusual long serum half-life in humans of nearly 3 weeks, which is attributed to its interaction with the neonatal Fc receptor (FcRn). FcRn protects albumin from intracellular degradation via a pH-dependent cellular recycling mechanism. To understand how FcRn impacts the role of albumin as a distributor, it is of importance to unravel the structural mechanism that determines pH-dependent binding. Here, we show that although the C-terminal domain III (DIII) of human serum albumin (HSA) contains the principal binding site, the N-terminal domain I (DI) is important for optimal FcRn binding. Specifically, structural inspection of human FcRn (hFcRn) in complex with HSA revealed that two exposed loops of DI were in proximity with the receptor. To investigate to what extent these contacts affected hFcRn binding, we targeted selected amino acid residues of the loops by mutagenesis. Screening by in vitro interaction assays revealed that several of the engineered HSA variants showed decreased binding to hFcRn, which was also the case for two missense variants with mutations within these loops. In addition, four of the variants showed improved binding. Our findings demonstrate that both DI and DIII are required for optimal binding to FcRn, which has implications for our understanding of the FcRn-albumin relationship and how albumin acts as a distributor. Such knowledge may inspire development of novel HSA-based diagnostics and therapeutics.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Receptores Fc/metabolismo , Albumina Sérica/química , Albumina Sérica/metabolismo , Substituição de Aminoácidos , Ligação Competitiva , Humanos , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Ligação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Albumina Sérica/genéticaRESUMO
Cardiac sodium (Na(+))-calcium (Ca(2+)) exchanger 1 (NCX1) is central to the maintenance of normal Ca(2+) homeostasis and contraction. Studies indicate that the Ca(2+)-activated protease calpain cleaves NCX1. We hypothesized that calpain is an important regulator of NCX1 in response to pressure overload and aimed to identify molecular mechanisms and functional consequences of calpain binding and cleavage of NCX1 in the heart. NCX1 full-length protein and a 75-kDa NCX1 fragment along with calpain were up-regulated in aortic stenosis patients and rats with heart failure. Patients with coronary artery disease and sham-operated rats were used as controls. Calpain co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes and left ventricle lysate. Immunoprecipitations, pull-down experiments, and extensive use of peptide arrays indicated that calpain domain III anchored to the first Ca(2+) binding domain in NCX1, whereas the calpain catalytic region bound to the catenin-like domain in NCX1. The use of bioinformatics, mutational analyses, a substrate competitor peptide, and a specific NCX1-Met(369) antibody identified a novel calpain cleavage site at Met(369). Engineering NCX1-Met(369) into a tobacco etch virus protease cleavage site revealed that specific cleavage at Met(369) inhibited NCX1 activity (both forward and reverse mode). Finally, a short peptide fragment containing the NCX1-Met(369) cleavage site was modeled into the narrow active cleft of human calpain. Inhibition of NCX1 activity, such as we have observed here following calpain-induced NCX1 cleavage, might be beneficial in pathophysiological conditions where increased NCX1 activity contributes to cardiac dysfunction.
Assuntos
Estenose da Valva Aórtica/metabolismo , Calpaína/metabolismo , Insuficiência Cardíaca/metabolismo , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Idoso , Sequência de Aminoácidos , Animais , Estenose da Valva Aórtica/genética , Estenose da Valva Aórtica/patologia , Sítios de Ligação , Calpaína/genética , Feminino , Células HEK293 , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Ventrículos do Coração/patologia , Humanos , Masculino , Dados de Sequência Molecular , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/patologia , Cultura Primária de Células , Ligação Proteica , Proteólise , Ratos , Ratos Wistar , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/genéticaRESUMO
Albumin is the most abundant protein in blood and plays a pivotal role as a multitransporter of a wide range of molecules such as fatty acids, metabolites, hormones, and toxins. In addition, it binds a variety of drugs. Its role as distributor is supported by its extraordinary serum half-life of 3 weeks. This is related to its size and binding to the cellular receptor FcRn, which rescues albumin from intracellular degradation. Furthermore, the long half-life has fostered a great and increasing interest in utilization of albumin as a carrier of protein therapeutics and chemical drugs. However, to fully understand how FcRn acts as a regulator of albumin homeostasis and to take advantage of the FcRn-albumin interaction in drug design, the interaction interface needs to be dissected. Here, we used a panel of monoclonal antibodies directed towards human FcRn in combination with site-directed mutagenesis and structural modeling to unmask the binding sites for albumin blocking antibodies and albumin on the receptor, which revealed that the interaction is not only strictly pH-dependent, but predominantly hydrophobic in nature. Specifically, we provide mechanistic evidence for a crucial role of a cluster of conserved tryptophan residues that expose a pH-sensitive loop of FcRn, and identify structural differences in proximity to these hot spot residues that explain divergent cross-species binding properties of FcRn. Our findings expand our knowledge of how FcRn is controlling albumin homeostasis at a molecular level, which will guide design and engineering of novel albumin variants with altered transport properties.
Assuntos
Albuminas/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Receptores Fc/química , Sequência de Aminoácidos , Anticorpos Bloqueadores/imunologia , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Receptores Fc/genética , Receptores Fc/imunologia , Receptores Fc/metabolismoRESUMO
A major challenge for the therapeutic use of many peptides and proteins is their short circulatory half-life. Albumin has an extended serum half-life of 3 weeks because of its size and FcRn-mediated recycling that prevents intracellular degradation, properties shared with IgG antibodies. Engineering the strictly pH-dependent IgG-FcRn interaction is known to extend IgG half-life. However, this principle has not been extensively explored for albumin. We have engineered human albumin by introducing single point mutations in the C-terminal end that generated a panel of variants with greatly improved affinities for FcRn. One variant (K573P) with 12-fold improved affinity showed extended serum half-life in normal mice, mice transgenic for human FcRn, and cynomolgus monkeys. Importantly, favorable binding to FcRn was maintained when a single-chain fragment variable antibody was genetically fused to either the N- or the C-terminal end. The engineered albumin variants may be attractive for improving the serum half-life of biopharmaceuticals.
Assuntos
Albuminas/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Receptores Fc/metabolismo , Albuminas/genética , Albuminas/farmacologia , Substituição de Aminoácidos , Animais , Feminino , Meia-Vida , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/farmacologia , Humanos , Macaca fascicularis , Camundongos , Mutação de Sentido Incorreto , Receptores Fc/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
The human 8-oxoguanine DNA glycosylase OGG1 is involved in base excision repair (BER), one of several DNA repair mechanisms that may counteract the effects of chemo- and radiation therapy for the treatment of cancer. We envisage that potent inhibitors of OGG1 may be found among the 9-alkyl-8-oxoguanines. Thus we explored synthetic routes to 8-oxoguanines and examined these as OGG1 inhibitors. The best reaction sequence started from 6-chloroguanine and involved N-9 alkylation, C-8 bromination, and finally simultaneous hydrolysis of both halides. Bromination before N-alkylation should only be considered when the N-substituent is not compatible with bromination conditions. The 8-oxoguanines were found to be weak inhibitors of OGG1. 6-Chloro-8-oxopurines, byproducts in the hydrolysis of 2,6-halopurines, turned out to be slightly better inhibitors than the corresponding 8-oxoguanines.
Assuntos
DNA Glicosilases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Guanina/análogos & derivados , Alquilação , DNA Glicosilases/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Guanina/síntese química , Guanina/química , Guanina/farmacologia , Humanos , Especificidade por SubstratoRESUMO
Stormorken syndrome is a rare autosomal-dominant disease with mild bleeding tendency, thrombocytopathy, thrombocytopenia, mild anemia, asplenia, tubular aggregate myopathy, miosis, headache, and ichthyosis. A heterozygous missense mutation in STIM1 exon 7 (c.910C>T; p.Arg304Trp) (NM_003156.3) was found to segregate with the disease in six Stormorken syndrome patients in four families. Upon sensing Ca(2+) depletion in the endoplasmic reticulum lumen, STIM1 undergoes a conformational change enabling it to interact with and open ORAI1, a Ca(2+) release-activated Ca(2+) channel located in the plasma membrane. The STIM1 mutation found in Stormorken syndrome patients is located in the coiled-coil 1 domain, which might play a role in keeping STIM1 inactive. In agreement with a possible gain-of-function mutation in STIM1, blood platelets from patients were in a preactivated state with high exposure of aminophospholipids on the outer surface of the plasma membrane. Resting Ca(2+) levels were elevated in platelets from the patients compared with controls, and store-operated Ca(2+) entry was markedly attenuated, further supporting constitutive activity of STIM1 and ORAI1. Thus, our data are compatible with a near-maximal activation of STIM1 in Stormorken syndrome patients. We conclude that the heterozygous mutation c.910C>T causes the complex phenotype that defines this syndrome.
Assuntos
Transtornos Plaquetários/genética , Dislexia/genética , Exoma/genética , Ictiose/genética , Proteínas de Membrana/genética , Transtornos de Enxaqueca/genética , Miose/genética , Mutação de Sentido Incorreto/genética , Proteínas de Neoplasias/genética , Baço/anormalidades , Adulto , Transtornos Plaquetários/patologia , Canais de Cálcio/genética , Hibridização Genômica Comparativa , Dislexia/patologia , Eritrócitos Anormais/patologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ictiose/patologia , Masculino , Transtornos de Enxaqueca/patologia , Miose/patologia , Fadiga Muscular/genética , Proteína ORAI1 , Linhagem , Baço/patologia , Molécula 1 de Interação EstromalRESUMO
The recently discovered HEAT-like repeat (HLR) DNA glycosylase superfamily is widely distributed in all domains of life. The present bioinformatics and phylogenetic analysis shows that HLR DNA glycosylase superfamily members in the genus Bacillus form three subfamilies: AlkC, AlkD and AlkF/AlkG. The crystal structure of AlkF shows structural similarity with the DNA glycosylases AlkC and AlkD, however neither AlkF nor AlkG display any DNA glycosylase activity. Instead, both proteins have affinity to branched DNA structures such as three-way and Holliday junctions. A unique ß-hairpin in the AlkF/AlkG subfamily is most likely inserted into the DNA major groove, and could be a structural determinant regulating DNA substrate affinity. We conclude that AlkF and AlkG represent a new family of HLR proteins with affinity for branched DNA structures.