RESUMO
Internal friction is a major contribution to the dynamics of intrinsically disordered proteins (IDPs). Yet, the molecular origin of internal friction has so far been elusive. Here, we investigate whether attractive electrostatic interactions in IDPs modulate internal friction differently than the hydrophobic effect. To this end, we used nanosecond fluorescence correlation spectroscopy (nsFCS) and single-molecule Förster resonance energy transfer (FRET) to quantify the conformation and dynamics of the disordered DNA-binding domains Myc, Max and Mad at different salt concentrations. We find that internal friction effects are stronger when the chain is compacted by electrostatic attractions compared to the hydrophobic effect. Although the effect is moderate, the results show that the heteropolymeric nature of IDPs is reflected in their dynamics.
Assuntos
Proteínas Intrinsicamente Desordenadas , Eletricidade Estática , Transferência Ressonante de Energia de Fluorescência , Fricção , PolímerosRESUMO
Dynamin oligomerizes into helical filaments on tubular membrane templates and, through constriction, cleaves them in a GTPase-driven way. Structural observations of GTP-dependent cross-bridges between neighboring filament turns have led to the suggestion that dynamin operates as a molecular ratchet motor. However, the proof of such mechanism remains absent. Particularly, it is not known whether a powerful enough stroke is produced and how the motor modules would cooperate in the constriction process. Here, we characterized the dynamin motor modules by single-molecule Förster resonance energy transfer (smFRET) and found strong nucleotide-dependent conformational preferences. Integrating smFRET with molecular dynamics simulations allowed us to estimate the forces generated in a power stroke. Subsequently, the quantitative force data and the measured kinetics of the GTPase cycle were incorporated into a model including both a dynamin filament, with explicit motor cross-bridges, and a realistic deformable membrane template. In our simulations, collective constriction of the membrane by dynamin motor modules, based on the ratchet mechanism, is directly reproduced and analyzed. Functional parallels between the dynamin system and actomyosin in the muscle are seen. Through concerted action of the motors, tight membrane constriction to the hemifission radius can be reached. Our experimental and computational study provides an example of how collective motor action in megadalton molecular assemblies can be approached and explicitly resolved.
Assuntos
Dinaminas/metabolismo , Modelos Biológicos , Fenômenos Biomecânicos , Dinaminas/química , Transferência Ressonante de Energia de Fluorescência , Cinética , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Nucleotídeos/metabolismo , Domínios Proteicos , Multimerização Proteica , SoluçõesRESUMO
Structural disorder is widespread in regulatory protein networks. Weak and transient interactions render disordered proteins particularly sensitive to fluctuations in solution conditions such as ion and crowder concentrations. How this sensitivity alters folding coupled binding reactions, however, has not been fully understood. Here, we demonstrate that salt jointly modulates polymer properties and binding affinities of 5 disordered proteins from a transcription factor network. A combination of single-molecule Förster resonance energy transfer experiments, polymer theory, and molecular simulations shows that all 5 proteins expand with increasing ionic strengths due to Debye-Hückel charge screening. Simultaneously, pairwise affinities between the proteins increase by an order of magnitude within physiological salt limits. A quantitative analysis shows that 50% of the affinity increase can be explained by changes in the disordered state. Disordered state properties therefore have a functional relevance even if these states are not directly involved in biological functions. Numerical solutions of coupled binding equilibria with our results show that networks of homologous disordered proteins can function surprisingly robustly in fluctuating cellular environments, despite the sensitivity of its individual proteins.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Polímeros/química , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Fatores de Transcrição de Zíper de Leucina Básica , Fenômenos Biofísicos , Transferência Ressonante de Energia de Fluorescência/métodos , Modelos Moleculares , Oócitos/metabolismo , Polímeros/metabolismo , Ligação Proteica/fisiologia , Conformação Proteica , Dobramento de Proteína , Proteínas Proto-Oncogênicas c-myc , Proteína Smad1 , Proteínas de Xenopus , Xenopus laevisRESUMO
Quantifying the stability of intermediates along parallel molecular pathways is often hampered by the limited experimental resolution of ensemble methods. In biology, however, such intermediates may represent important regulatory targets, thus calling for strategies to map their abundance directly. Here, we use single-molecule Förster resonance energy transfer (FRET) to quantify the occupancies of intermediates along two parallel DNA-binding pathways of the basic helix-loop-helix leucine-zipper (bHLH-LZ) domains of the transcription factors c-Myc and Max. We find that both proteins are intrinsically disordered with sub-microsecond end-to-end distance dynamics. In mixtures of the proteins with their promoter DNA, our experiments identify the disordered conformers, the folded Myc-Max dimer, and ternary Myc-Max-DNA complexes. However, signatures of the intermediate along the alternative pathway, i.e., one domain bound to DNA, remained undetectable. This implies that disordered Max-DNA and Myc-DNA complexes are by at least 6 kB T higher in free energy than folded dimers of Myc and Max. The disordered monomer-DNA complex is therefore unlikely to be of importance for the regulation of transcriptional processes.
Assuntos
DNA/química , Proteínas Intrinsicamente Desordenadas/química , Sítios de Ligação , Transferência Ressonante de Energia de Fluorescência , Sequências Hélice-Alça-Hélice , Zíper de LeucinaRESUMO
Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein implicated in the pathogenesis of both familial and sporadic Parkinson's disease (PD), and currently one of the most promising therapeutic targets for drug design in Parkinson's disease. In contrast, LRRK1, the closest homologue to LRRK2, does not play any role in PD. Here, we use cryo-electron microscopy (cryo-EM) and single particle analysis to gain structural insight into the full-length dimeric structures of LRRK2 and LRRK1. Differential scanning fluorimetry-based screening of purification buffers showed that elution of the purified LRRK2 protein in a high pH buffer is beneficial in obtaining high quality cryo-EM images. Next, analysis of the 3D maps generated from the cryo-EM data show 16 and 25 Å resolution structures of full length LRRK2 and LRRK1, respectively, revealing the overall shape of the dimers with two-fold symmetric orientations of the protomers that is closely similar between the two proteins. These results suggest that dimerization mechanisms of both LRRKs are closely related and hence that specificities in functions of each LRRK are likely derived from LRRK2 and LRRK1's other biochemical functions. To our knowledge, this study is the first to provide 3D structural insights in LRRK2 and LRRK1 dimers in parallel.
Assuntos
Microscopia Crioeletrônica , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Soluções Tampão , Detergentes/farmacologia , Humanos , Imageamento Tridimensional , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Dobramento de Proteína/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , SoluçõesRESUMO
The range of ATP concentrations that can be measured with a fluorescent reagentless biosensor for ATP has been increased by modulating its affinity for this analyte. The ATP biosensor is an adduct of two tetramethylrhodamines with MatB from Rhodopseudomonas palustris. Mutations were introduced into the binding site to modify ATP binding affinity, while aiming to maintain the concomitant fluorescence signal. Using this signal, the effect of mutations in different parts of the binding site was measured. This mutational analysis revealed three variants in particular, each with a single mutation in the phosphate-binding loop, which had potentially beneficial changes in ATP binding properties but preserving a fluorescence change of ~3-fold on ATP binding. Two variants (T167A and T303A) weakened the binding, changing the dissociation constant from the parent's 6 µM to 123 µM and 42 µM, respectively. Kinetic measurements showed that the effect of these mutations on affinity was by an increase in dissociation rate constants. These variants widen the range of ATP concentration that can be measured readily by this biosensor to >100 µM. In contrast, a third variant, S170A, decreased the dissociation constant of ATP to 3.8 µM and has a fluorescence change of 4.2 on binding ATP. This variant has increased selectivity for ATP over ADP of >200-fold. This had advantages over the parent by increasing sensitivity as well as increasing selectivity during ATP measurements in which ADP is present.
Assuntos
Trifosfato de Adenosina/análise , Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais , Coenzima A Ligases/metabolismo , Corantes Fluorescentes/química , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Coenzima A Ligases/química , Coenzima A Ligases/genética , Cinética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Piruvato Quinase/química , Piruvato Quinase/metabolismo , Rodaminas/química , Rodopseudomonas/enzimologiaRESUMO
A fluorescent reagentless biosensor for ATP has been developed, based on malonyl-coenzyme A synthetase from Rhodopseudomonas palustris as the protein scaffold and recognition element. Two 5-iodoacetamidotetramethylrhodamines were covalently bound to this protein to provide the readout. This adduct couples ATP binding to a 3.7-fold increase in fluorescence intensity with excitation at 553 nm and emission at 575 nm. It measures ATP concentrations with micromolar sensitivity and is highly selective for ATP relative to ADP. Its ability to monitor enzymatic ATP production or depletion was demonstrated in steady-state kinetic assays in which ATP is a product or substrate, respectively.
Assuntos
Trifosfato de Adenosina/análise , Proteínas de Bactérias/química , Técnicas Biossensoriais , Coenzima A Ligases/química , Corantes Fluorescentes/química , Trifosfato de Adenosina/química , Proteínas de Bactérias/metabolismo , Coenzima A Ligases/metabolismo , Modelos Biológicos , Piruvato Quinase/química , Piruvato Quinase/farmacologiaRESUMO
Leucine-rich repeat kinase 2 (LRRK2) is a complex, multidomain protein which is considered a valuable target for potential disease-modifying therapeutic strategies for Parkinson's disease (PD). In mammalian cells and brain, LRRK2 is phosphorylated and treatment of cells with inhibitors of LRRK2 kinase activity can induce LRRK2 dephosphorylation at a cluster of serines including Ser910/935/955/973. It has been suggested that phosphorylation levels at these sites reflect LRRK2 kinase activity, however kinase-dead variants of LRRK2 or kinase activating variants do not display altered Ser935 phosphorylation levels compared to wild type. Furthermore, Ser910/935/955/973 are not autophosphorylation sites, therefore, it is unclear if inhibitor induced dephosphorylation depends on the activity of compounds on LRRK2 or on yet to be identified upstream kinases. Here we used a panel of 160 ATP competitive and cell permeable kinase inhibitors directed against all branches of the kinome and tested their activity on LRRK2 in vitro using a peptide-substrate-based kinase assay. In neuronal SH-SY5Y cells overexpressing LRRK2 we used compound-induced dephosphorylation of Ser935 as readout. In silico docking of selected compounds was performed using a modeled LRRK2 kinase structure. Receiver operating characteristic plots demonstrated that the obtained docking scores to the LRRK2 ATP binding site correlated with in vitro and cellular compound activity. We also found that in vitro potency showed a high degree of correlation to cellular compound induced LRRK2 dephosphorylation activity across multiple compound classes. Therefore, acute LRRK2 dephosphorylation at Ser935 in inhibitor treated cells involves a strong component of inhibitor activity on LRRK2 itself, without excluding a role for upstream kinases. Understanding the regulation of LRRK2 phosphorylation by kinase inhibitors aids our understanding of LRRK2 signaling and may lead to development of new classes of LRRK2 kinase inhibitors.
RESUMO
Recent genome-wide association studies indicate that a simple alteration of Leucine-rich repeat kinase 2 (LRRK2) gene expression may contribute to the etiology of sporadic Parkinson's disease (PD). However, the expression and regulation of LRRK2 protein in the sporadic PD brains remain to be determined. Here, we found that the expression of LRRK2 protein was enhanced in the sporadic PD patients using the frontal cortex tissue from a set of 16 PD patients and 7 control samples. In contrast, no significant difference was detected in the level of LRRK2 mRNA expression between the control and PD cases, suggesting a potential post-transcriptional modification of the LRRK2 protein expression in the sporadic PD brains. Indeed, it was identified that microRNA-205 (miR-205) suppressed the expression of LRRK2 protein through a conserved-binding site at the 3'-untranslated region (UTR) of LRRK2 gene. Interestingly, miR-205 expression was significantly downregulated in the brains of patients with sporadic PD, showing the enhanced LRRK2 protein levels. Also, in vitro studies in the cell lines and primary neuron cultures further established the role of miR-205 in modulating the expression of LRRK2 protein. In addition, introduction of miR-205 prevented the neurite outgrowth defects in the neurons expressing a PD-related LRRK2 R1441G mutant. Together, these findings suggest that downregulation of miR-205 may contribute to the potential pathogenic elevation of LRRK2 protein in the brains of patients with sporadic PD, while overexpression of miR-205 may provide an applicable therapeutic strategy to suppress the abnormal upregulation of LRRK2 protein in PD.
Assuntos
MicroRNAs/metabolismo , Doença de Parkinson/genética , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Encéfalo/patologia , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Regulação para Baixo , Marcadores Genéticos , Estudo de Associação Genômica Ampla , Células HEK293 , Células HeLa , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Neurônios/citologia , Neurônios/metabolismo , Doença de Parkinson/fisiopatologia , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/fisiopatologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Regulação para CimaRESUMO
Leucine-rich repeat kinase 1 and 2 (LRRK1 and LRRK2) are large multidomain proteins containing kinase, GTPase and multiple protein-protein interaction domains, but only mutations in LRRK2 are linked to familial Parkinson's disease (PD). Independent studies suggest that LRRK2 exists in the cell as a complex compatible with the size of a dimer. However, whether this complex is truly a homodimer or a heterologous complex formed by monomeric LRRK2 with other proteins has not been definitively proven due to the limitations in obtaining highly pure proteins suitable for structural characterization. Here, we used stable expression of LRRK1 and LRRK2 in HEK293T cell lines to produce recombinant LRRK1 and LRRK2 proteins of greater than 90% purity. Both purified LRRKs are folded, with a predominantly alpha-helical secondary structure and are capable of binding GTP with similar affinity. Furthermore, recombinant LRRK2 exhibits robust autophosphorylation activity, phosphorylation of model peptides in vitro and ATP binding. In contrast, LRRK1 does not display significant autophosphorylation activity and fails to phosphorylate LRRK2 model substrates, although it does bind ATP. Using these biochemically validated proteins, we show that LRRK1 and LRRK2 are capable of forming homodimers as shown by single-particle transmission electron microscopy and immunogold labeling. These LRRK dimers display an elongated conformation with a mean particle size of 145 Å and 175 Å respectively, which is disrupted by addition of 6M guanidinium chloride. Immunogold staining revealed double-labeled particles also in the pathological LRRK2 mutant G2019S and artificial mutants disrupting GTPase and kinase activities, suggesting that point mutations do not hinder the dimeric conformation. Overall, our findings indicate for the first time that purified and active LRRK1 and LRRK2 can form dimers in their full-length conformation.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/química , Trifosfato de Adenosina/química , Cromatografia/métodos , Dicroísmo Circular , Dimerização , Células HEK293 , Humanos , Imuno-Histoquímica , Lentivirus/genética , Proteínas de Repetições Ricas em Leucina , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Fluorescência/métodos , Mutação , Fosforilação , Mutação Puntual , Ligação Proteica , Proteínas Serina-Treonina Quinases/químicaRESUMO
DNA binding as well as ligand binding by nuclear receptors has been studied extensively. Both binding functions are attributed to isolated domains of which the structure is known. The crystal structure of a complete receptor in complex with its ligand and DNA-response element, however, has been solved only for the peroxisome proliferator-activated receptor γ (PPARγ)-retinoid X receptor α (RXRα) heterodimer. This structure provided the first indication of direct interactions between the DNA-binding domain (DBD) and ligand-binding domain (LBD). In this study, we investigated whether there is a similar interface between the DNA- and ligand-binding domains for the androgen receptor (AR). Despite the structural differences between the AR- and PPARγ-LBD, a combination of in silico modeling and docking pointed out a putative interface between AR-DBD and AR-LBD. The surfaces were subjected to a point mutation analysis, which was inspired by known AR mutations described in androgen insensitivity syndromes and prostate cancer. Surprisingly, AR-LBD mutations D695N, R710A, F754S, and P766A induced a decrease in DNA binding but left ligand binding unaffected, while the DBD-residing mutations K590A, K592A, and E621A lowered the ligand-binding but not the DNA-binding affinity. We therefore propose that these residues are involved in allosteric communications between the AR-DBD and AR-LBD.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Receptores Androgênicos/química , Receptores Androgênicos/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Cristalização , Proteínas de Ligação a DNA/genética , Ligantes , Modelos Moleculares , Mutação , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores Androgênicos/genéticaRESUMO
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. Much research effort has been directed towards the catalytic core region of LRRK2 composed of GTPase (ROC, Ras of complex proteins) and kinase domains and a connecting COR (C-terminus of ROC) domain. In contrast, the precise functions of the protein-protein interaction domains, such as the leucine-rich repeat (LRR) domain, are not known. In the present study, we modeled the LRRK2 LRR domain (LRR(LRRK2)) using a template assembly approach, revealing the presence of 14 LRRs. Next, we focused on the expression and purification of LRR(LRRK2) in Escherichia coli. Buffer optimization revealed that the protein requires the presence of a zwitterionic detergent, namely Empigen BB, during solubilization and the subsequent purification and characterization steps. This indicates that the detergent captures the hydrophobic surface patches of LRR(LRRK2) thereby suppressing its aggregation. Circular dichroism (CD) spectroscopy measured 18% α-helices and 21% ß-sheets, consistent with predictions from the homology model. Size exclusion chromatography (SEC) and dynamic light scattering measurements showed the presence of a single species, with a Stokes radius corresponding to the model dimensions of a protein monomer. Furthermore, no obvious LRR(LRRK2) multimerization was detected via cross-linking studies. Finally, the LRR(LRRK2) clinical mutations did not influence LRR(LRRK2) secondary, tertiary or quaternary structure as determined via SEC and CD spectroscopy. We therefore conclude that these mutations are likely to affect putative LRR(LRRK2) inter- and intramolecular interactions.
Assuntos
Proteínas Serina-Treonina Quinases/química , Proteínas Recombinantes/química , Sequência de Aminoácidos , Cromatografia em Gel , Dicroísmo Circular , Escherichia coli/genética , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Luz , Dados de Sequência Molecular , Mutação , Compostos Orgânicos , Doença de Parkinson/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espalhamento de Radiação , Alinhamento de SequênciaRESUMO
Intense research efforts are currently directed at elucidating the etiology of Parkinson's disease (PD). One approach that has begun to shed light on the PD pathogenic pathways is the identification of disease genes through genetic linkage or association studies. These studies have revealed that several kinases may be involved in PD, as some PD genes encode kinases themselves while other PD genes are found in the same cellular pathways as kinases. Two of these kinases stand out as potential drug targets for novel PD therapy, namely leucine rich repeat kinase 2 (LRRK2) and the alpha-synuclein (α-syn) phosphorylating polo-like kinase 2 (PLK2). Indeed, both α- syn and LRRK2 show genetic linkage as well as genetic association with PD, indicating their relevance to a large number of PD cases. Also, due to the dominant mode of α-syn and LRRK2 inheritance and based on current knowledge of LRRK2 and α-syn phosphorylation by PLK2, inhibition of LRRK2 and PLK2 may constitute a potential therapy for PD. Here we discuss the function of these kinases as well as progress in their validation as drug targets for the treatment of PD.
Assuntos
Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Doença de Parkinson/enzimologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , alfa-Sinucleína/antagonistas & inibidores , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Leucine rich repeat kinase 2 (LRRK2) is a Parkinson's disease (PD) gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound "on" state and a GDP bound "off" state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC) GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Substituição de Aminoácidos , Sítios de Ligação/genética , GTP Fosfo-Hidrolases/genética , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/análogos & derivados , Células HEK293 , Humanos , Immunoblotting , Imunoprecipitação , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Mutação , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: Transportin-SR2 (TRN-SR2, TNPO3, transportin 3) was previously identified as an interaction partner of human immunodeficiency virus type 1 (HIV-1) integrase and functions as a nuclear import factor of HIV-1. A possible role of capsid in transportin-SR2-mediated nuclear import was recently suggested by the findings that a chimeric HIV virus, carrying the murine leukemia virus (MLV) capsid and matrix proteins, displayed a transportin-SR2 independent phenotype, and that the HIV-1 N74D capsid mutant proved insensitive to transportin-SR2 knockdown. RESULTS: Our present analysis of viral specificity reveals that TRN-SR2 is not used to the same extent by all lentiviruses. The DNA flap does not determine the TRN-SR2 requirement of HIV-1. We corroborate the TRN-SR2 independent phenotype of the chimeric HIV virus carrying the MLV capsid and matrix proteins. We reanalyzed the HIV-1 N74D capsid mutant in cells transiently or stably depleted of transportin-SR2 and confirm that the N74D capsid mutant is independent of TRN-SR2 when pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). Remarkably, although somewhat less dependent on TRN-SR2 than wild type virus, the N74D capsid mutant carrying the wild type HIV-1 envelope required TRN-SR2 for efficient replication. By pseudotyping with envelopes that mediate pH-independent viral uptake including HIV-1, measles virus and amphotropic MLV envelopes, we demonstrate that HIV-1 N74D capsid mutant viruses retain partial dependency on TRN-SR2. However, this dependency on TRN-SR2 is lost when the HIV N74D capsid mutant is pseudotyped with envelopes mediating pH-dependent endocytosis, such as the VSV-G and Ebola virus envelopes. CONCLUSION: Here we discover a link between the viral entry of HIV and its interaction with TRN-SR2. Our data confirm the importance of TRN-SR2 in HIV-1 replication and argue for careful interpretation of experiments performed with VSV-G pseudotyped viruses in studies on early steps of HIV replication including the role of capsid therein.
Assuntos
HIV-1/fisiologia , Internalização do Vírus , beta Carioferinas/metabolismo , Linhagem Celular , HumanosRESUMO
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent known cause of autosomal dominant Parkinson's disease. The LRRK2 gene encodes a Roco protein featuring a Ras of complex proteins (ROC) GTPase and a kinase domain linked by the C-terminal of ROC (COR) domain. Here, we explored the effects of the Y1699C pathogenic LRRK2 mutation in the COR domain on GTPase activity and interactions within the catalytic core of LRRK2. We observed a decrease in GTPase activity for LRRK2 Y1699C comparable to the decrease observed for the R1441C pathogenic mutant and the T1348N dysfunctional mutant. To study the underlying mechanism, we explored the dimerization in the catalytic core of LRRK2. ROC-COR dimerization was significantly weakened by the Y1699C or R1441C/G mutation. Using a competition assay, we demonstrated that the intra-molecular ROC : COR interaction is favoured over ROC : ROC dimerization. Interestingly, the intra-molecular ROC : COR interaction was strengthened by the Y1699C mutation. This is supported by a 3D homology model of the ROC-COR tandem of LRRK2, showing that Y1699 is positioned at the intra-molecular ROC : COR interface. In conclusion, our data provides mechanistic insight into the mode of action of the Y1699C LRRK2 mutant: the Y1699C substitution, situated at the intra-molecular ROC : COR interface, strengthens the intra-molecular ROC : COR interaction, thereby locally weakening the dimerization of LRRK2 at the ROC-COR tandem domain resulting in decreased GTPase activity.
Assuntos
Mutação , Proteínas Serina-Treonina Quinases/genética , Domínio Catalítico/genética , Cisteína/genética , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Ligação Proteica/genética , Multimerização Proteica/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Tirosina/genéticaRESUMO
Hereditary thrombotic thrombocytopenic purpura is caused by mutations in a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS13) resulting in defective processing of von Willebrand factor (VWF) that causes intravascular platelet aggregation culminating in thrombocytopenia with shistocytic anemia. In this study the functional and structural role of a recently identified ADAMTS13 metalloprotease domain mutation S119F was investigated. Secretion from heterologous cells was hampered but not completely eliminated. Secreted S119F was active toward multimeric VWF and FRETS-VWF73 but with abnormal kinetics, having a significantly reduced overall catalytic rate (k(cat); 0.88 +/- 0.04 s(-1) vs 2.78 +/- 0.11 s(-1)) and slightly smaller Michaelis constant (K(M); 1.4 +/- 0.2microM vs 2.3 +/- 0.3microM). A computational model of the metalloprotease domain demonstrates both steric and polar interaction effects caused by S119F. Interestingly, mutant S119A has properties similar to S119F (k(cat) = 0.82 +/- 0.03 s(-1) and K(M) = 1.1 +/- 0.1microM), allowing to assign distorted kinetics to the loss of the H-bond with conserved residue W262. We conclude that the S119-W262 H-bond is crucial for maximal turnover.
Assuntos
Proteínas ADAM/química , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Púrpura Trombocitopênica Trombótica/genética , Púrpura Trombocitopênica Trombótica/metabolismo , Proteína ADAMTS13 , Adolescente , Western Blotting , Humanos , Masculino , Mutação , Estrutura Quaternária de ProteínaRESUMO
Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited Parkinson's disease (PD). The protein is large and complex, but pathogenic mutations cluster in a region containing GTPase and kinase domains. LRRK2 can autophosphorylate in vitro within a dimer pair, although the significance of this reaction is unclear. Here, we mapped the sites of autophosphorylation within LRRK2 and found several potential phosphorylation sites within the GTPase domain. Using mass spectrometry, we found that Thr1343 is phosphorylated and, using kinase dead versions of LRRK2, show that this is an autophosphorylation site. However, we also find evidence for additional sites in the GTPase domain and in other regions of the protein suggesting that there may be multiple autophosphorylation sites within LRRK2. These data suggest that the kinase and GTPase activities of LRRK2 may exhibit complex autoregulatory interdependence.