RESUMO
Two coding variants of apolipoprotein L1 (APOL1), called G1 and G2, explain much of the excess risk of kidney disease in African Americans. While various cytotoxic phenotypes have been reported in experimental models, the proximal mechanism by which G1 and G2 cause kidney disease is poorly understood. Here, we leveraged 3 experimental models and a recently reported small molecule blocker of APOL1 protein, VX-147, to identify the upstream mechanism of G1-induced cytotoxicity. In HEK293 cells, we demonstrated that G1-mediated Na+ import/K+ efflux triggered activation of GPCR/IP3-mediated calcium release from the ER, impaired mitochondrial ATP production, and impaired translation, which were all reversed by VX-147. In human urine-derived podocyte-like epithelial cells (HUPECs), we demonstrated that G1 caused cytotoxicity that was again reversible by VX-147. Finally, in podocytes isolated from APOL1 G1 transgenic mice, we showed that IFN-γ-mediated induction of G1 caused K+ efflux, activation of GPCR/IP3 signaling, and inhibition of translation, podocyte injury, and proteinuria, all reversed by VX-147. Together, these results establish APOL1-mediated Na+/K+ transport as the proximal driver of APOL1-mediated kidney disease.
Assuntos
Apolipoproteína L1 , Nefropatias , Compostos Organotiofosforados , Camundongos , Animais , Humanos , Apolipoproteína L1/genética , Células HEK293 , Variação Genética , Nefropatias/genética , Camundongos TransgênicosRESUMO
Disruption in copper homeostasis causes a number of cognitive and motor deficits. Wilson's disease and Menkes disease are neurodevelopmental disorders resulting from mutations in the copper transporters ATP7A and ATP7B, with ATP7A mutations also causing occipital horn syndrome, and distal motor neuropathy. A 65 year old male presenting with brachial amyotrophic diplegia and diagnosed with amyotrophic lateral sclerosis (ALS) was found to harbor a p.Met1311Val (M1311V) substitution variant in ATP7A. ALS is a fatal neurodegenerative disease associated with progressive muscle weakness, synaptic deficits and degeneration of upper and lower motor neurons. To investigate the potential contribution of the ATP7AM1311V variant to neurodegeneration, we obtained and characterized both patient-derived fibroblasts and patient-derived induced pluripotent stem cells differentiated into motor neurons (iPSC-MNs), and compared them to control cell lines. We found reduced localization of ATP7AM1311V to the trans-Golgi network (TGN) at basal copper levels in patient-derived fibroblasts and iPSC-MNs. In addition, redistribution of ATP7AM1311V out of the TGN in response to increased extracellular copper was defective in patient fibroblasts. This manifested in enhanced intracellular copper accumulation and reduced survival of ATP7AM1311V fibroblasts. iPSC-MNs harboring the ATP7AM1311V variant showed decreased dendritic complexity, aberrant spontaneous firing, and decreased survival. Finally, expression of the ATP7AM1311V variant in Drosophila motor neurons resulted in motor deficits. Apilimod, a drug that targets vesicular transport and recently shown to enhance survival of C9orf72-ALS/FTD iPSC-MNs, also increased survival of ATP7AM1311V iPSC-MNs and reduced motor deficits in Drosophila expressing ATP7AM1311V. Taken together, these observations suggest that ATP7AM1311V negatively impacts its role as a copper transporter and impairs several aspects of motor neuron function and morphology.
Assuntos
ATPases Transportadoras de Cobre/genética , ATPases Transportadoras de Cobre/metabolismo , Cobre/metabolismo , Variação Genética/fisiologia , Doença dos Neurônios Motores/genética , Doença dos Neurônios Motores/metabolismo , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Células Cultivadas , Cobre/farmacologia , Cobre/uso terapêutico , Relação Dose-Resposta a Droga , Drosophila , Variação Genética/efeitos dos fármacos , Células HeLa , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Doença dos Neurônios Motores/tratamento farmacológico , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologiaRESUMO
BACKGROUND: Two coding renal risk variants (RRVs) of the APOL1 gene (G1 and G2) are associated with large increases in CKD rates among populations of recent African descent, but the underlying molecular mechanisms are unknown. Mammalian cell culture models are widely used to study cytotoxicity of RRVs, but results have been contradictory. It remains unclear whether cytotoxicity is RRV-dependent or driven solely by variant-independent overexpression. It is also unknown whether expression of the reference APOL1 allele, the wild-type G0, could prevent cytotoxicity of RRVs. METHODS: We generated tetracycline-inducible APOL1 expression in human embryonic kidney HEK293 cells and examined the effects of increased expression of APOL1 (G0, G1, G2, G0G0, G0G1, or G0G2) on known cytotoxicity phenotypes, including reduced viability, increased swelling, potassium loss, aberrant protein phosphorylation, and dysregulated energy metabolism. Furthermore, whole-genome transcriptome analysis examined deregulated canonical pathways. RESULTS: At moderate expression, RRVs but not G0 caused cytotoxicity in a dose-dependent manner that coexpression of G0 did not reduce. RRVs also have dominant effects on canonical pathways relevant for the cellular stress response. CONCLUSIONS: In HEK293 cells, RRVs exhibit a dominant toxic gain-of-function phenotype that worsens with increasing expression. These observations suggest that high steady-state levels of RRVs may underlie cellular injury in APOL1 nephropathy, and that interventions that reduce RRV expression in kidney compartments may mitigate APOL1 nephropathy.
Assuntos
Apolipoproteína L1/genética , Apolipoproteína L1/fisiologia , Sobrevivência Celular , Metabolismo Energético , Perfilação da Expressão Gênica , Variação Genética , Células HEK293 , Humanos , Potássio/metabolismo , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
Two specific genetic variants of the apolipoprotein L1 (APOL1) gene are responsible for the high rate of kidney disease in people of recent African ancestry. Expression in cultured cells of these APOL1 risk variants, commonly referred to as G1 and G2, results in significant cytotoxicity. The underlying mechanism of this cytotoxicity is poorly understood. We hypothesized that this cytotoxicity is mediated by APOL1 risk variant-induced dysregulation of intracellular signaling relevant for cell survival. To test this hypothesis, we conditionally expressed WT human APOL1 (G0), the APOL1 G1 variant, or the APOL1 G2 variant in human embryonic kidney cells (T-REx-293) using a tetracycline-mediated (Tet-On) system. We found that expression of either G1 or G2 APOL1 variants increased apparent cell swelling and cell death compared with G0-expressing cells. These manifestations of cytotoxicity were preceded by G1 or G2 APOL1-induced net efflux of intracellular potassium as measured by X-ray fluorescence, resulting in the activation of stress-activated protein kinases (SAPKs), p38 MAPK, and JNK. Prevention of net K(+) efflux inhibited activation of these SAPKs by APOL1 G1 or G2. Furthermore, inhibition of SAPK signaling and inhibition of net K(+) efflux abrogated cytotoxicity associated with expression of APOL1 risk variants. These findings in cell culture raise the possibility that nephrotoxicity of APOL1 risk variants may be mediated by APOL1 risk variant-induced net loss of intracellular K(+) and subsequent induction of stress-activated protein kinase pathways.
Assuntos
Apolipoproteínas/genética , Transporte de Íons/genética , Nefropatias/genética , Lipoproteínas HDL/genética , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Mutação de Sentido Incorreto , Potássio/metabolismo , Substituição de Aminoácidos , Apolipoproteína L1 , Apolipoproteínas/fisiologia , População Negra/genética , Morte Celular , Tamanho Celular , Receptor gp130 de Citocina/biossíntese , Receptor gp130 de Citocina/genética , Progressão da Doença , Ativação Enzimática , Frequência do Gene , Predisposição Genética para Doença , Células HEK293 , Humanos , Nefropatias/etnologia , Lipoproteínas HDL/fisiologia , Sistema de Sinalização das MAP Quinases , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/metabolismo , Risco , Fator de Transcrição STAT3/metabolismo , TransfecçãoRESUMO
N-Substituted glycine peptoid oligomers have recently attracted attention for their metal binding capabilities. Due to their efficient synthesis on solid phase, peptoids are well suited for generation of compound libraries, followed by screening for molecular recognition and other diverse functional attributes. Ideally, peptoids could be simultaneously screened for binding to a number of metal species. Here, we demonstrate the use of bench-top X-ray fluorescence (XRF) instrumentation to screen rapidly, on solid support, a library of peptoid oligomers incorporating metal-binding functionalities. A subset of the peptoid sequences exhibited significant metal binding capabilities, including a peptoid pentamer and a nonamer that were shown to selectively bind nickel. The binding capabilities were validated by colorimetric assay and by depletion of Ni(2+) ion concentration from solution, establishing bench-top XRF as a rapid, practicable high-throughput screening technique for peptoid oligomers. This protocol will facilitate discovery of metallopeptoids with unique material properties.
Assuntos
Metais/metabolismo , Peptoides/metabolismo , Resinas Sintéticas/química , Espectrometria por Raios X , Colorimetria , Níquel , Oximas/química , Biblioteca de Peptídeos , SoluçõesRESUMO
Understanding the complex biochemical mechanisms that underlie the regulation, toxicity, and protein binding of metal ions requires the ability to analyze the metal content of individual proteins in complex mixtures. In this issue of ACS Chemical Biology, a technique combining gel electrophoresis with synchrotron X-ray fluorescence imaging demonstrates a rapid and powerful solution for simultaneously examining multiple proteins and metal ions of interest. The resulting technique is broadly applicable, does not require specialized equipment for sample preparation, and is likely to be extensible in the future.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Metais/isolamento & purificação , Proteínas/isolamento & purificação , Eletroforese em Gel de Poliacrilamida/economia , Fluorescência , Raios XRESUMO
Bacterial ribonuclease P (RNase P) catalyzes the cleavage of 5' leader sequences from precursor tRNAs (pre-tRNAs). Previously, all known substrate nucleotide specificities in this system are derived from RNA-RNA interactions with the RNase P RNA subunit. Here, we demonstrate that pre-tRNA binding affinities for Bacillus subtilis and Escherichia coli RNase P are enhanced by sequence-specific contacts between the fourth pre-tRNA nucleotide on the 5' side of the cleavage site (N(-4)) and the RNase P protein (P protein) subunit. B. subtilis RNase P has a higher affinity for pre-tRNA with adenosine at N(-4), and this binding preference is amplified at physiological divalent ion concentrations. Measurements of pre-tRNA-containing adenosine analogs at N(-4) indicate that specificity arises from a combination of hydrogen bonding to the N6 exocyclic amine of adenosine and steric exclusion of the N2 amine of guanosine. Mutagenesis of B. subtilis P protein indicates that F20 and Y34 contribute to selectivity at N(-4). The hydroxyl group of Y34 enhances selectivity, likely by forming a hydrogen bond with the N(-4) nucleotide. The sequence preference of E. coli RNase P is diminished, showing a weak preference for adenosine and cytosine at N(-4), consistent with the substitution of Leu for Y34 in the E. coli P protein. This is the first identification of a sequence-specific contact between P protein and pre-tRNA that contributes to molecular recognition of RNase P. Additionally, sequence analyses reveal that a greater-than-expected fraction of pre-tRNAs from both E. coli and B. subtilis contains a nucleotide at N(-4) that enhances RNase P affinity. This observation suggests that specificity at N(-4) contributes to substrate recognition in vivo. Furthermore, bioinformatic analyses suggest that sequence-specific contacts between the protein subunit and the leader sequences of pre-tRNAs may be common in bacterial RNase P and may lead to species-specific substrate recognition.
Assuntos
Regiões 5' não Traduzidas/genética , Bacillus subtilis/enzimologia , Escherichia coli/enzimologia , Precursores de RNA/metabolismo , Ribonuclease P/metabolismo , Adenosina/metabolismo , Substituição de Aminoácidos/efeitos dos fármacos , Bacillus subtilis/genética , Sequência de Bases , Cálcio/farmacologia , Escherichia coli/genética , Genoma Bacteriano , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Nucleotídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , RNA de Transferência/genética , Ribonuclease P/química , Especificidade por Substrato/efeitos dos fármacosRESUMO
Bacterial ribonuclease P (RNase P) is a ribonucleoprotein complex composed of one catalytic RNA (PRNA) and one protein subunit (P protein) that together catalyze the 5' maturation of precursor tRNA. High-resolution X-ray crystal structures of the individual P protein and PRNA components from several species have been determined, and structural models of the RNase P holoenzyme have been proposed. However, holoenzyme models have been limited by a lack of distance constraints between P protein and PRNA in the holoenzyme-substrate complex. Here, we report the results of extensive cross-linking and affinity cleavage experiments using single-cysteine P protein variants derivatized with either azidophenacyl bromide or 5-iodoacetamido-1,10-o-phenanthroline to determine distance constraints and to model the Bacillus subtilis holoenzyme-substrate complex. These data indicate that the evolutionarily conserved RNR motif of P protein is located near (<15 Angstroms) the pre-tRNA cleavage site, the base of the pre-tRNA acceptor stem and helix P4 of PRNA, the putative active site of the enzyme. In addition, the metal binding loop and N-terminal region of the P protein are proximal to the P3 stem-loop of PRNA. Studies using heterologous holoenzymes composed of covalently modified B. subtilis P protein and Escherichia coli M1 RNA indicate that P protein binds similarly to both RNAs. Together, these data indicate that P protein is positioned close to the RNase P active site and may play a role in organizing the RNase P active site.
Assuntos
Bacillus subtilis/enzimologia , Reagentes de Ligações Cruzadas/metabolismo , RNA Bacteriano , RNA Catalítico , Ribonuclease P/química , Ribonuclease P/metabolismo , Substituição de Aminoácidos , Bacillus subtilis/genética , Sequência de Bases , Sítios de Ligação , Catálise , Domínio Catalítico , Cisteína/genética , Escherichia coli/química , Holoenzimas/química , Holoenzimas/metabolismo , Radical Hidroxila/química , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Catalítico/química , RNA Catalítico/genética , RNA Catalítico/metabolismoRESUMO
The ribonucleoprotein enzyme RNase P processes all pre-tRNAs, yet some substrates apparently lack consensus elements for recognition. Here, we compare binding affinities and cleavage rates of Escherichia coli pre-tRNAs that exhibit the largest variation from consensus recognition sequences. These results reveal that the affinities of both consensus and nonconsensus substrates for the RNase P holoenzyme are essentially uniform. Comparative analyses of pre-tRNA and tRNA binding to the RNase P holoenzyme and P RNA alone reveal differential contributions of the protein subunit to 5' leader and tRNA affinity. Additionally, these studies reveal that uniform binding results from variations in the energetic contribution of the 5' leader, which serve to compensate for weaker tRNA interactions. Furthermore, kinetic analyses reveal uniformity in the rates of substrate cleavage that result from dramatic (> 900-fold) contributions of the protein subunit to catalysis for some nonconsensus pre-tRNAs. Together, these data suggest that an important biological function of RNase P protein is to offset differences in pre-tRNA structure such that binding and catalysis are uniform.
Assuntos
Proteínas de Escherichia coli/química , Ribonuclease P/química , Região 5'-Flanqueadora , Sequência de Bases , Sítios de Ligação , Catálise , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Precursores de RNA/química , RNA Bacteriano/química , RNA de Transferência/química , Especificidade por SubstratoRESUMO
Fidelity in tRNA processing by the RNase P RNA from Escherichia coli depends, in part, on interactions with the nucleobase and 2' hydroxyl group of N(-1), the nucleotide immediately upstream of the site of RNA strand cleavage. Here, we report a series of biochemical and structure-function studies designed to address how these interactions contribute to cleavage site selection. We find that simultaneous disruption of cleavage site nucleobase and 2' hydroxyl interactions results in parallel reactions leading to correct cleavage and mis-cleavage one nucleotide upstream (5') of the correct site. Changes in Mg(2+) concentration and pH can influence the fraction of product that is incorrectly processed, with pH effects attributable to differences in the rate-limiting steps for the correct and mis-cleavage reaction pathways. Additionally, we provide evidence that interactions with the 2' hydroxyl group adjacent to the reactive phosphate group also contribute to catalysis at the mis-cleavage site. Finally, disruption of the adjacent 2'-hydroxyl contact has a greater effect on catalysis when pairing between the ribozyme and N(-1) is also disrupted, and the effects of simultaneously disrupting these contacts on binding are also non-additive. One implication of these results is that mis-cleavage will result from any combination of active site modifications that decrease the rate of correct cleavage beyond a certain threshold. Indeed, we find that inhibition of correct cleavage and corresponding mis-cleavage also results from disruption of any combination of active site contacts including metal ion interactions and conserved pairing interactions with the 3' RCCA sequence. Such redundancy in interactions needed for maintaining fidelity may reflect the necessity for multiple substrate recognition in vivo. These studies provide a framework for interpreting effects of substrate modifications on RNase P cleavage fidelity and provide evidence for interactions with the nucleobase and 2' hydroxyl group adjacent to the reactive phosphate group in the transition state.
Assuntos
Nucleotídeos/metabolismo , Processamento Pós-Transcricional do RNA , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribonuclease P/metabolismo , Pareamento de Bases , Sítios de Ligação , Catálise , Concentração de Íons de Hidrogênio , Hidroxilação , Cinética , Magnésio/farmacologia , Mutação/genética , Fosfatos/metabolismo , RNA de Transferência/genética , Ribonuclease P/genéticaRESUMO
The bacterial tRNA processing enzyme ribonuclease P (RNase P) is a ribonucleoprotein composed of a approximately 400 nucleotide RNA and a smaller protein subunit. It has been established that RNase P RNA contacts the mature tRNA portion of pre-tRNA substrates, whereas RNase P protein interacts with the 5' leader sequence. However, specific interactions with substrate nucleotides flanking the cleavage site have not previously been defined. Here we provide evidence for an interaction between a conserved adenosine, A248 in the Escherichia coli ribozyme, and N(-1), the substrate nucleotide immediately 5' of the cleavage site. Specifically, mutations at A248 result in miscleavage of substrates containing a 2' deoxy modification at N(-1). Compensatory mutations at N(-1) restore correct cleavage in both the RNA-alone and holoenzyme reactions, and also rescue defects in binding thermodynamics caused by A248 mutation. Analysis of pre-tRNA leader sequences in Bacteria and Archaea reveals a conserved preference for U at N(-1), suggesting that an interaction between A248 and N(-1) is common among RNase P enzymes. These results provide the first direct evidence for RNase P RNA interactions with the substrate cleavage site, and show that RNA and protein cooperate in leader sequence recognition.
Assuntos
Regiões 5' não Traduzidas/metabolismo , Endorribonucleases/química , Endorribonucleases/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Precursores de RNA/metabolismo , RNA Catalítico/química , RNA Catalítico/metabolismo , RNA de Transferência/metabolismo , Adenosina/genética , Sequência de Bases , Sítios de Ligação , Catálise , Sequência Conservada , Escherichia coli/enzimologia , Modelos Biológicos , Mutação , Precursores de RNA/química , RNA Arqueal/química , RNA Arqueal/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA de Transferência/química , Ribonuclease P , Análise de Sequência de RNA , Especificidade por SubstratoRESUMO
Ribonuclease P (RNase P), is a ribonucleoprotein complex that catalyzes the site-specific cleavage of pre-tRNA and a wide variety of other substrates. Although RNase P RNA is the catalytic subunit of the holoenzyme, the protein subunit plays a critical role in substrate binding. Thus, RNase P is an excellent model system for studying ribonucleoprotein function. In this review we describe methods applied to the in vitro study of substrate recognition by bacterial RNase P, covering general considerations of reaction conditions, quantitative measurement of substrate binding equilibria, enzymatic and chemical protection, cross-linking, modification interference, and analysis of site-specific substitutions. We describe application of these methods to substrate binding by RNase P RNA alone and experimental considerations for examining the holoenzyme. The combined use of these approaches has shown that the RNA and protein subunits cooperate to bind different portions of the substrate structure, with the RNA subunit predominantly interacting with the mature domain of tRNA and the protein interacting with the 5(') leader sequence. However, important questions concerning the interface between the two subunits and the coordination of RNA and protein subunits in binding and catalysis remain.
Assuntos
Endorribonucleases/metabolismo , Precursores de RNA/metabolismo , RNA Catalítico/metabolismo , Endorribonucleases/química , Conformação de Ácido Nucleico , Ligação Proteica , RNA Catalítico/química , Ribonuclease P , Especificidade por SubstratoRESUMO
Like protein enzymes, catalytic RNAs contain conserved structure motifs important for function. A universal feature of the catalytic domain of ribonuclease P RNA is a bulged-helix motif within the P1-P4 helix junction. Here, we show that changes in bulged nucleotide identity and position within helix P4 affect both catalysis and substrate binding, while a subset of the mutations resulted only in catalytic defects. We find that the proximity of the bulge to sites of metal ion coordination in P4 is important for catalysis; moving the bulge distal to these sites and deleting it had similarly large effects, while moving it proximal to these sites had only a moderate effect on catalysis. To test whether the effects of the mutations are linked to metal ion interactions, we used terbium-dependent cleavage of the phosphate backbone to probe metal ion-binding sites in the wild-type and mutant ribozymes. We detect cleavages at specific sites within the catalytic domain, including helix P4 and J3/4, which have previously been shown to participate directly in metal ion interactions. Mutations introduced into P4 cause local changes in the terbium cleavage pattern due to alternate metal ion-binding configurations with the helix. In addition, a bulge deletion mutation results in a 100-fold decrease in the single turnover cleavage rate constant at saturating magnesium levels, and a reduced affinity for magnesium ions important for catalysis. In light of the alternate terbium cleavage pattern in P4 caused by bulge deletion, this decreased ability to utilize magnesium ions for catalysis appears to be due to localized structural changes in the ribozyme's catalytic core that weaken metal ion interactions in P4 and J3/4. The information reported here, therefore, provides evidence that the universal conservation of the P4 structure is based in part on optimization of metal ion interactions important for catalysis.