RESUMO
The DNA double-strand breaks (DSBs) that initiate meiotic recombination are formed by an evolutionarily conserved suite of factors that includes Rec114 and Mei4 (RM), which regulate DSB formation both spatially and temporally. In vivo, these proteins form large immunostaining foci that are integrated with higher-order chromosome structures. In vitro, they form a 2:1 heterotrimeric complex that binds cooperatively to DNA to form large, dynamic condensates. However, understanding of the atomic structures and dynamic DNA binding properties of RM complexes is lacking. Here, we report a structural model of a heterotrimeric complex of the C terminus of Rec114 with the N terminus of Mei4, supported by nuclear magnetic resonance experiments. This minimal complex, which lacks the predicted intrinsically disordered region of Rec114, is sufficient to bind DNA and form condensates. Single-molecule experiments reveal that the minimal complex can bridge two or more DNA duplexes and can generate force to condense DNA through long-range interactions. AlphaFold2 predicts similar structural models for RM orthologs across diverse taxa despite their low degree of sequence similarity. These findings provide insight into the conserved networks of protein-protein and protein-DNA interactions that enable condensate formation and promote formation of meiotic DSBs.
Assuntos
Proteínas de Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Cromossomos/metabolismo , Meiose , Quebras de DNA de Cadeia Dupla , DNARESUMO
G protein-coupled receptors (GPCRs) control intracellular signaling cascades via agonist-dependent coupling to intracellular transducers including heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. In addition to their critical interactions with the transmembrane core of active GPCRs, all three classes of transducers have also been reported to interact with receptor C-terminal domains (CTDs). An underexplored aspect of GPCR CTDs is their possible role as lipid sensors given their proximity to the membrane. CTD-membrane interactions have the potential to control the accessibility of key regulatory CTD residues to downstream effectors and transducers. Here, we report that the CTDs of two closely related family C GPCRs, metabotropic glutamate receptor 2 (mGluR2) and mGluR3, bind to membranes and that this interaction can regulate receptor function. We first characterize CTD structure with NMR spectroscopy, revealing lipid composition-dependent modes of membrane binding. Using molecular dynamics simulations and structure-guided mutagenesis, we then identify key conserved residues and cancer-associated mutations that modulate CTD-membrane binding. Finally, we provide evidence that mGluR3 transducer coupling is controlled by CTD-membrane interactions in live cells, which may be subject to regulation by CTD phosphorylation and changes in membrane composition. This work reveals an additional mechanism of GPCR modulation, suggesting that CTD-membrane binding may be a general regulatory mode throughout the broad GPCR superfamily.
Assuntos
Membrana Celular , Simulação de Dinâmica Molecular , Receptores de Glutamato Metabotrópico , Humanos , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/genética , Membrana Celular/metabolismo , Domínios Proteicos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/química , Ligação Proteica , Células HEK293 , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Transdução de SinaisRESUMO
Post-translationally modified tau is the primary component of tau neurofibrillary tangles, a pathological hallmark of Alzheimer's disease and other tauopathies. Post-translational modifications (PTMs) within the tau microtubule (MT)-binding domain (MBD), which encompasses two hexapeptide motifs that act as critical nucleating regions for tau aggregation, can potentially modulate tau aggregation as well as interactions with MTs and membranes. Here, we characterize the effects of a recently discovered tau PTM, lysine succinylation, on tau-tubulin interactions and compare these to the effects of two previously reported MBD modifications, lysine acetylation and tyrosine phosphorylation. As generation of site-specific PTMs in proteins is challenging, we used short synthetic peptides to quantify the effects on tubulin binding of three site-specific PTMs located within the PHF6∗ (paired helical filament [PHF] residues 275-280) and PHF6 (residues 306-311) hexapeptide motifs: K280 acetylation, Y310 phosphorylation, and K311 succinylation. We compared these effects to those observed for MBD PTM-mimetic point mutations K280Q, Y310E, and K311E. Finally, we evaluated the effects of these PTM-mimetic mutations on MBD membrane binding and membrane-induced fibril and oligomer formation. We found that all three PTMs perturb tau MT binding, with Y310 phosphorylation exerting the strongest effect. PTM-mimetic mutations partially recapitulated the effects of the PTMs on MT binding and also disrupted tau membrane binding and membrane-induced oligomer and fibril formation. These results imply that these PTMs, including the novel and Alzheimer's disease-specific succinylation of tau K311, may influence both the physiological and pathological interactions of tau and thus represent targets for therapeutic intervention.
Assuntos
Doença de Alzheimer , Microtúbulos , Emaranhados Neurofibrilares , Processamento de Proteína Pós-Traducional , Proteínas tau , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Humanos , Lisina/metabolismo , Microtúbulos/metabolismo , Emaranhados Neurofibrilares/metabolismo , Fosforilação , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteínas tau/metabolismoRESUMO
Alpha-synuclein (a-Syn) is a presynaptic protein, the misfolding of which is associated with Parkinson's disease. Rab GTPases are small guanine nucleotide binding proteins that play key roles in vesicle trafficking and have been associated with a-Syn function and dysfunction. a-Syn is enriched on synaptic vesicles, where it has been reported to interact with GTP-bound Rab3a, a master regulator of synaptic vesicle trafficking. a-Syn is known to bind weakly to Rab8a in solution via a positively charged patch, but the physiological implications of such interactions have not been explored. Here, we investigate direct interactions between a-Syn and Rab3a in solution and on lipid membranes using NMR spectroscopy. We find that the C terminus of a-Syn interacts with Rab3a in a manner similar to its previously reported interaction with Rab8a. While weak in solution, we demonstrate that this interaction becomes stronger when the proteins are bound to a membrane surface. The Rab3a binding site for a-Syn is similar to the surface that contacts the Rab3a effector rabphilin-3A, which modulates the enzymatic activity of Rab3a. Accordingly, we show that a-Syn inhibits GTP hydrolysis by Rab3a and that inhibition is more potent on the membrane surface, suggesting that their interaction may be functionally relevant. Finally, we show that phosphorylation of a-Syn residue Ser 129, a modification associated with Parkinson's disease pathology, enhances its interactions with Rab3a and increases its ability to inhibit Rab3a GTP hydrolysis. These results represent the first observation of a functional role for synuclein-Rab interactions and for a-Syn Ser 129 phosphorylation.
Assuntos
Doença de Parkinson , alfa-Sinucleína , Proteína rab3A de Ligação ao GTP , Guanosina Trifosfato/metabolismo , Humanos , Lipídeos/química , Doença de Parkinson/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Proteína rab3A de Ligação ao GTP/química , Proteína rab3A de Ligação ao GTP/genética , Proteína rab3A de Ligação ao GTP/metabolismoRESUMO
Limited chemical shift dispersion represents a significant barrier to studying multistate equilibria of large membrane proteins by 19F NMR. We describe a novel monofluoroethyl 19F probe that dramatically increases the chemical shift dispersion. The improved conformational sensitivity and line shape enable the detection of previously unresolved states in one-dimensional (1D) 19F NMR spectra of a 134 kDa membrane transporter. Changes in the populations of these states in response to ligand binding, mutations, and temperature correlate with population changes of distinct conformations in structural ensembles determined by single-particle cryo-electron microscopy (cryo-EM). Thus, 19F NMR can guide sample preparation to discover and visualize novel conformational states and facilitate image analysis and three-dimensional (3D) classification.
Assuntos
Flúor , Imageamento por Ressonância Magnética , Microscopia Crioeletrônica/métodos , Espectroscopia de Ressonância Magnética , Conformação ProteicaRESUMO
In proteins where conformational changes are functionally important, the number of accessible states and their dynamics are often difficult to establish. Here we describe a novel 19F-NMR spectroscopy approach to probe dynamics of large membrane proteins. We labeled a glutamate transporter homolog with a 19F probe via cysteine chemistry and with a Ni2+ ion via chelation by a di-histidine motif. We used distance-dependent enhancement of the longitudinal relaxation of 19F nuclei by the paramagnetic metal to assign the observed resonances. We identified one inward- and two outward-facing states of the transporter, in which the substrate-binding site is near the extracellular and intracellular solutions, respectively. We then resolved the structure of the unanticipated second outward-facing state by cryo-EM. Finally, we showed that the rates of the conformational exchange are accessible from measurements of the metal-enhanced longitudinal relaxation of 19F nuclei.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/química , Espectroscopia de Ressonância Magnética , Sistema X-AG de Transporte de Aminoácidos/genética , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Microscopia Crioeletrônica , Cisteína/química , Flúor , Histidina/química , Modelos Moleculares , Mutação , Níquel/química , Conformação Proteica , Domínios Proteicos , Pyrococcus horikoshii/químicaRESUMO
The microtubule-associated protein Tau is implicated in the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease. Increasing evidence suggests that post-translational modifications play critical roles in regulating Tau's normal functions and its pathogenic properties in tauopathies. Very little is known about how phosphorylation of tyrosine residues influences the structure, aggregation, and microtubule- and lipid-binding properties of Tau. Here, we sought to determine the relative contributions of phosphorylation of one or several of the five tyrosine residues in Tau (Tyr-18, -29, -197, -310, and -394) to the regulation of its biophysical, aggregation, and functional properties. We used a combination of site-specific mutagenesis and in vitro phosphorylation by c-Abl kinase to generate Tau species phosphorylated at all five tyrosine residues, all tyrosine residues except Tyr-310 or Tyr-394 (pTau-Y310F and pTau-Y394F, respectively) and Tau phosphorylated only at Tyr-310 or Tyr-394 (4F/pTyr-310 or 4F/pTyr-394). We observed that phosphorylation of all five tyrosine residues, multiple N-terminal tyrosine residues (Tyr-18, -29, and -197), or specific phosphorylation only at residue Tyr-310 abolishes Tau aggregation and inhibits its microtubule- and lipid-binding properties. NMR experiments indicated that these effects are mediated by a local decrease in ß-sheet propensity of Tau's PHF6 domain. Our findings underscore Tyr-310 phosphorylation has a unique role in the regulation of Tau aggregation, microtubule, and lipid interactions. These results also highlight the importance of conducting further studies to elucidate the role of Tyr-310 in the regulation of Tau's normal functions and pathogenic properties.
Assuntos
Lipídeos/química , Microtúbulos/metabolismo , Tirosina/metabolismo , Proteínas tau/metabolismo , Sítios de Ligação , Humanos , Microtúbulos/química , Fosforilação , Tirosina/química , Proteínas tau/químicaRESUMO
Post-translational modifications (PTMs) can affect the normal function and pathology of α-synuclein (αS), an amyloid-fibril-forming protein linked to Parkinson's disease. Phosphorylation of αS Tyr39 has recently been found to display a dose-dependent effect on fibril formation kinetics and to alter the morphology of the fibrils. Existing methods to access site-specifically phosphorylated αS for biochemical studies include total or semi-synthesis by native chemical ligation (NCL) as well as chemoenzymatic methods to phosphorylate peptides, followed by NCL. Here, we investigated a streamlined method to produce large quantities of phosphorylated αS by co-expressing a kinase with a protein fragment in Escherichia coli. We also introduced the use of methyl thioglycolate (MTG) to enable one-pot NCL and desulfurization. We compare our optimized methods to previous reports and show that we can achieve the highest yields of site-specifically phosphorylated protein through chemoenzymatic methods using MTG, and that our strategy is uniquely well suited to producing 15 N-labeled, phosphorylated protein for NMR studies.
Assuntos
Tirosina/metabolismo , alfa-Sinucleína/biossíntese , Estrutura Molecular , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Tirosina/química , alfa-Sinucleína/químicaRESUMO
The past few years have resulted in an increased awareness and recognition of the prevalence and roles of intrinsically disordered proteins and protein regions (IDPs and IDRs, respectively) in synaptic vesicle trafficking and exocytosis and in overall synaptic organization. IDPs and IDRs constitute a class of proteins and protein regions that lack stable tertiary structure, but nevertheless retain biological function. Their significance in processes such as cell signaling is now well accepted, but their pervasiveness and importance in other areas of biology are not as widely appreciated. Here, we review the prevalence and functional roles of IDPs and IDRs associated with the release and recycling of synaptic vesicles at nerve terminals, as well as with the architecture of these terminals. We hope to promote awareness, especially among neuroscientists, of the importance of this class of proteins in these critical pathways and structures. The examples discussed illustrate some of the ways in which the structural flexibility conferred by intrinsic protein disorder can be functionally advantageous in the context of cellular trafficking and synaptic function.
Assuntos
Exocitose/fisiologia , Proteínas Intrinsicamente Desordenadas/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Transporte Biológico Ativo/fisiologia , HumanosRESUMO
Prion-like transcellular spreading of tau in Alzheimer's Disease (AD) is mediated by tau binding to cell surface heparan sulfate (HS). However, the structural determinants for tau-HS interaction are not well understood. Microarray and SPR assays of structurally defined HS oligosaccharides show that a rare 3-O-sulfation (3-O-S) of HS significantly enhances tau binding. In Hs3st1-/- (HS 3-O-sulfotransferase-1 knockout) cells, reduced 3-O-S levels of HS diminished both cell surface binding and internalization of tau. In a cell culture, the addition of a 3-O-S HS 12-mer reduced both tau cell surface binding and cellular uptake. NMR titrations mapped 3-O-S binding sites to the microtubule binding repeat 2 (R2) and proline-rich region 2 (PRR2) of tau. Tau is only the seventh protein currently known to recognize HS 3-O-sulfation. Our work demonstrates that this rare 3-O-sulfation enhances tau-HS binding and likely the transcellular spread of tau, providing a novel target for disease-modifying treatment of AD and other tauopathies.
Assuntos
Doença de Alzheimer/genética , Membrana Celular/metabolismo , Heparitina Sulfato/química , Proteínas tau/metabolismo , Células Cultivadas , HumanosRESUMO
The StARkin superfamily comprises proteins with steroidogenic acute regulatory protein-related lipid transfer (StART) domains that are implicated in intracellular, non-vesicular lipid transport. A new family of membrane-anchored StARkins was recently identified, including six members, Lam1-Lam6, in the yeast Saccharomyces cerevisiae. Lam1-Lam4 are anchored to the endoplasmic reticulum (ER) membrane at sites where the ER is tethered to the plasma membrane and proposed to be involved in sterol homeostasis in yeast. To better understand the biological roles of these proteins, we carried out a structure-function analysis of the second StARkin domain of Lam4, here termed Lam4S2. NMR experiments indicated that Lam4S2 undergoes specific conformational changes upon binding sterol, and fluorescence-based assays revealed that it catalyzes sterol transport between vesicle populations in vitro, exhibiting a preference for vesicles containing anionic lipids. Using such vesicles, we found that sterols are transported at a rate of â¼50 molecules per Lam4S2 per minute. Crystal structures of Lam4S2, with and without bound sterol, revealed a largely hydrophobic but surprisingly accessible sterol-binding pocket with the 3-OH group of the sterol oriented toward its base. Single or multiple alanine or aspartic acid replacements of conserved lysine residues in a basic patch on the surface of Lam4S2 near the likely sterol entry/egress site strongly attenuated sterol transport. Our results suggest that Lam4S2 engages anionic membranes via a basic surface patch, enabling "head-first" entry of sterol into the binding pocket followed by partial closure of the entryway. Reversal of these steps enables sterol egress.
Assuntos
Antiporters/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Esteróis/química , Antiporters/genética , Antiporters/metabolismo , Transporte Biológico Ativo/fisiologia , Domínios Proteicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esteróis/metabolismoRESUMO
Fibrillar α-synuclein (AS) is the major component of Lewy bodies, the pathological hallmark of Parkinson's disease. Using solid-state nuclear magnetic resonance (ssNMR), we previously reported a structural characterization of mouse AS (mAS) fibrils and found that the secondary structure of the mAS fibrils is highly similar to a form of human AS (hAS) fibrils. Recently, a three-dimensional structure of these same hAS fibrils was determined by ssNMR and scanning transmission electron microscopy. Using medium- and long-range distance restraints obtained from ssNMR spectra, we found that the single protofilament structure of mAS fibrils is also similar to that of the hAS fibrils. However, residue-specific water accessibility of mAS fibrils probed by water polarization transfer ssNMR measurements indicates that residues S42-T44 and G84-V95 are largely protected from water even though they are located at the edge of the protofilament. Some of the corresponding resonances also exhibit peak doubling. These observations suggest that these residues may be involved in, to our knowledge, a novel protofilament-protofilament interface. We propose a structural model of mAS fibrils that incorporates this dimer interface.
Assuntos
Agregados Proteicos , alfa-Sinucleína/química , Animais , Camundongos , Estrutura Secundária de Proteína , Água/químicaRESUMO
The dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is believed to play a central role in Parkinson's disease neurodegeneration by stabilizing potentially toxic oligomers of the presynaptic protein α-Synuclein (aSyn). Besides the formation of covalent DOPAL-Lys adducts, DOPAL promotes the oxidation of Met residues of aSyn, which is also a common oxidative post-translational modification found in the protein in vivo. Herein we set out to address the role of Met residues on the oligomerization and neurotoxic properties of DOPAL-modified aSyn. Our data indicate that DOPAL promotes the formation of two distinct types of aSyn oligomers: large and small (dimer and trimers) oligomers, which seem to be generated by independent mechanisms and cannot be interconverted by using denaturing agents. Interestingly, H2O2-treated aSyn monomer, which exhibits all-four Met residues oxidized to Met-sulfoxide, exhibited a reduced ability to form large oligomers upon treatment with DOPAL, with no effect on the population of small oligomers. In this context, triple Met-Val mutant M5V/M116V/M127V exhibited an increased population of large aSyn-DOPAL oligomers in comparison with the wild-type protein. Interestingly, the stabilization of large rather than small oligomers seems to be associated with an enhanced toxicity of DOPAL-aSyn adducts. Collectively, these findings indicate that Met residues may play an important role in modulating both the oligomerization and the neurotoxic properties of DOPAL-derived aSyn species.
Assuntos
Ácido 3,4-Di-Hidroxifenilacético/farmacologia , Metionina/química , Neurônios/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , alfa-Sinucleína/toxicidade , Ácido 3,4-Di-Hidroxifenilacético/análogos & derivados , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Peróxido de Hidrogênio/farmacologia , Metionina/genética , Camundongos , Mutação , Neurônios/citologia , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMO
Tau aggregates into paired helical filaments within neurons, a pathological hallmark of Alzheimer's disease. Heparin promotes tau aggregation and recently has been shown to be involved in the cellular uptake of tau aggregates. Although the tau-heparin interaction has been extensively studied, little is known about the glycan determinants of this interaction. Here, we used surface plasmon resonance (SPR) and NMR spectroscopy to characterize the interaction between two tau fragments, K18 and K19, and several polysaccharides, including heparin, heparin oligosaccharides, chemically modified heparin, and related glycans. Using a heparin-immobilized chip, SPR revealed that tau K18 and K19 bind heparin with a KD of 0.2 and 70 µM, respectively. In SPR competition experiments, N-desulfation and 2-O-desulfation had no effect on heparin binding to K18, whereas 6-O-desulfation severely reduced binding, suggesting a critical role for 6-O-sulfation in the tau-heparin interaction. The tau-heparin interaction became stronger with longer-chain heparin oligosaccharides. As expected for an electrostatics-driven interaction, a moderate amount of salt (0.3 M NaCl) abolished binding. NMR showed the largest chemical-shift perturbation (CSP) in R2 in tau K18, which was absent in K19, revealing differential binding sites in K18 and K19 to heparin. Dermatan sulfate binding produced minimal CSP, whereas dermatan disulfate, with the additional 6-O-sulfo group, induced much larger CSP. 2-O-desulfated heparin induced much larger CSP in K18 than 6-O-desulfated heparin. Our data demonstrate a crucial role for the 6-O-sulfo group in the tau-heparin interaction, which to our knowledge has not been reported before.
Assuntos
Heparina/química , Heparina/metabolismo , Proteínas tau/metabolismo , Relação Dose-Resposta a Droga , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Cloreto de Sódio/farmacologia , Ressonância de Plasmônio de Superfície , Proteínas tau/químicaRESUMO
NEMO is the regulatory subunit of the IkappaB kinase (IKK) in NF-kappaB activation, and its CC2-LZ region interacts with Lys63 (K63)-linked polyubiquitin to recruit IKK to receptor signaling complexes. In vitro, CC2-LZ also interacts with tandem diubiquitin. Here we report the crystal structure of CC2-LZ with two dimeric coiled coils representing CC2 and LZ, respectively. Surprisingly, mutagenesis and nuclear magnetic resonance experiments reveal that the binding sites for diubiquitins at LZ are composites of both chains and that each ubiquitin in diubiquitins interacts with symmetrical NEMO asymmetrically. For tandem diubiquitin, the first ubiquitin uses the conserved hydrophobic patch and the C-terminal tail, while the second ubiquitin uses an adjacent surface patch. For K63-linked diubiquitin, the proximal ubiquitin uses its conserved hydrophobic patch, while the distal ubiquitin mostly employs the C-terminal arm including the K63 linkage residue. These studies uncover the energetics and geometry for mutual recognition of NEMO and diubiquitins.
Assuntos
Quinase I-kappa B/química , Ubiquitinas/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Sequência Conservada , Cristalografia por Raios X , Predisposição Genética para Doença , Humanos , Interações Hidrofóbicas e Hidrofílicas , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , NF-kappa B/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Relação Estrutura-Atividade , Ubiquitinas/metabolismoRESUMO
Oxidative deamination of dopamine produces the highly toxic aldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL), enhanced production of which is found in post-mortem brains of Parkinson disease patients. When injected into the substantia nigra of rat brains, DOPAL causes the loss of dopaminergic neurons accompanied by the accumulation of potentially toxic oligomers of the presynaptic protein α-synuclein (aS), potentially explaining the synergistic toxicity described for dopamine metabolism and aS aggregation. In this work, we demonstrate that DOPAL interacts with aS via formation of Schiff-base and Michael-addition adducts with Lys residues, in addition to causing oxidation of Met residues to Met-sulfoxide. DOPAL modification leads to the formation of small aS oligomers that may be cross-linked by DOPAL. Both monomeric and oligomeric DOPAL adducts potently inhibit the formation of mature amyloid fibrils by unmodified aS. The binding of aS to either lipid vesicles or detergent micelles, which results in a gain of α-helix structure in its N-terminal lipid-binding domain, protects the protein against DOPAL adduct formation and, consequently, inhibits DOPAL-induced aS oligomerization. Functionally, aS-DOPAL monomer exhibits a reduced affinity for small unilamellar vesicles with lipid composition similar to synaptic vesicles, in addition to diminished membrane-induced α-helical content in comparison with the unmodified protein. These results suggest that DOPAL could compromise the functionality of aS, even in the absence of protein oligomerization, by affecting the interaction of aS with lipid membranes and hence its role in the regulation of synaptic vesicle traffic in neurons.
Assuntos
Ácido 3,4-Di-Hidroxifenilacético/análogos & derivados , Amiloide/química , Dopamina/metabolismo , Lipídeos de Membrana/química , Doença de Parkinson/metabolismo , alfa-Sinucleína/química , Ácido 3,4-Di-Hidroxifenilacético/química , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/toxicidade , Amiloide/metabolismo , Animais , Membrana Celular/química , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Humanos , Lisina/química , Lipídeos de Membrana/metabolismo , Oxirredução , Doença de Parkinson/patologia , Ratos , Bases de Schiff/química , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo , Substância Negra/patologia , alfa-Sinucleína/metabolismoRESUMO
Increasing evidence suggests that the c-Abl protein tyrosine kinase could play a role in the pathogenesis of Parkinson's disease (PD) and other neurodegenerative disorders. c-Abl has been shown to regulate the degradation of two proteins implicated in the pathogenesis of PD, parkin and α-synuclein (α-syn). The inhibition of parkin's neuroprotective functions is regulated by c-Abl-mediated phosphorylation of parkin. However, the molecular mechanisms by which c-Abl activity regulates α-syn toxicity and clearance remain unknown. Herein, using NMR spectroscopy, mass spectrometry, in vitro enzymatic assays and cell-based studies, we established that α-syn is a bona fide substrate for c-Abl. In vitro studies demonstrate that c-Abl directly interacts with α-syn and catalyzes its phosphorylation mainly at tyrosine 39 (pY39) and to a lesser extent at tyrosine 125 (pY125). Analysis of human brain tissues showed that pY39 α-syn is detected in the brains of healthy individuals and those with PD. However, only c-Abl protein levels were found to be upregulated in PD brains. Interestingly, nilotinib, a specific inhibitor of c-Abl kinase activity, induces α-syn protein degradation via the autophagy and proteasome pathways, whereas the overexpression of α-syn in the rat midbrains enhances c-Abl expression. Together, these data suggest that changes in c-Abl expression, activation and/or c-Abl-mediated phosphorylation of Y39 play a role in regulating α-syn clearance and contribute to the pathogenesis of PD.
Assuntos
Doença de Parkinson/enzimologia , Proteínas Proto-Oncogênicas c-abl/metabolismo , alfa-Sinucleína/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/enzimologia , Encéfalo/metabolismo , Encéfalo/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fosforilação , Proteólise , Proteínas Proto-Oncogênicas c-abl/genética , alfa-Sinucleína/genéticaRESUMO
A novel mutation in the α-Synuclein (α-Syn) gene "G51D" was recently identified in two familial cases exhibiting features of Parkinson's disease (PD) and multiple system atrophy (MSA). In this study, we explored the impact of this novel mutation on the aggregation, cellular and biophysical properties of α-Syn, in an attempt to unravel how this mutant contributes to PD/MSA. Our results show that the G51D mutation significantly attenuates α-Syn aggregation in vitro. Moreover, it disrupts local helix formation in the presence of SDS, decreases binding to lipid vesicles C-terminal to the site of mutation and severely inhibits helical folding in the presence of acidic vesicles. When expressed in yeast, α-Syn(G51D) behaves similarly to α-Syn(A30P), as both exhibit impaired membrane association, form few inclusions and are non-toxic. In contrast, enhanced secreted and nuclear levels of the G51D mutant were observed in mammalian cells, as well as in primary neurons, where α-Syn(G51D) was enriched in the nuclear compartment, was hyper-phosphorylated at S129 and exacerbated α-Syn-induced mitochondrial fragmentation. Finally, post-mortem human brain tissues of α-Syn(G51D) cases were examined, and revealed only partial colocalization with nuclear membrane markers, probably due to post-mortem tissue delay and fixation. These findings suggest that the PD-linked mutations may cause neurodegeneration via different mechanisms, some of which may be independent of α-Syn aggregation.
Assuntos
Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Mutação/genética , Doença de Parkinson/genética , Agregação Patológica de Proteínas/genética , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Soluções Tampão , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Corpos de Inclusão/efeitos dos fármacos , Corpos de Inclusão/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neuroblastoma/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Membrana Nuclear/efeitos dos fármacos , Membrana Nuclear/metabolismo , Doença de Parkinson/patologia , Fosforilação/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Transporte Proteico/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Dodecilsulfato de Sódio/farmacologia , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Lipossomas Unilamelares/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/ultraestruturaRESUMO
The steroidogenic acute regulatory protein-related lipid transfer (START) domain family is defined by a conserved 210-amino acid sequence that folds into an α/ß helix-grip structure. Members of this protein family bind a variety of ligands, including cholesterol, phospholipids, sphingolipids, and bile acids, with putative roles in nonvesicular lipid transport, metabolism, and cell signaling. Among the soluble START proteins, STARD4 is expressed in most tissues and has previously been shown to transfer sterol, but the molecular mechanisms of membrane interaction and sterol binding remain unclear. In this work, we use biochemical techniques to characterize regions of STARD4 and determine their role in membrane interaction and sterol binding. Our results show that STARD4 interacts with anionic membranes through a surface-exposed basic patch and that introducing a mutation (L124D) into the Omega-1 (Ω1) loop, which covers the sterol binding pocket, attenuates sterol transfer activity. To gain insight into the attenuating mechanism of the L124D mutation, we conducted structural and biophysical studies of wild-type and L124D STARD4. These studies show that the L124D mutation reduces the conformational flexibility of the protein, resulting in a diminished level of membrane interaction and sterol transfer. These studies also reveal that the C-terminal α-helix, and not the Ω1 loop, partitions into the membrane bilayer. On the basis of these observations, we propose a model of STARD4 membrane interaction and sterol binding and release that requires dynamic movement of both the Ω1 loop and membrane insertion of the C-terminal α-helix.
Assuntos
Membrana Celular , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Simulação de Dinâmica Molecular , Esteróis/química , Esteróis/metabolismo , Substituição de Aminoácidos , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de Membrana Transportadoras/genética , Camundongos , Mutação de Sentido Incorreto , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The Parkinson disease protein α-synuclein is N-terminally acetylated, but most in vitro studies have been performed using unacetylated α-synuclein. Binding to lipid membranes is considered key to the still poorly understood function of α-synuclein. We report the effects of N-terminal acetylation on α-synuclein binding to lipid vesicles of different composition and curvature and to micelles composed of the detergents ß-octyl-glucoside (BOG) and SDS. In the presence of SDS, N-terminal acetylation results in a slightly increased helicity for the N-terminal ~10 residues of the protein, likely due to the stabilization of N-terminal fraying through the formation of a helix cap motif. In the presence of BOG, a detergent used in previous isolations of helical oligomeric forms of α-synuclein, the N-terminally acetylated protein adopts a novel conformation in which the N-terminal ~30 residues bind the detergent micelle in a partly helical conformation, whereas the remainder of the protein remains unbound and disordered. Binding of α-synuclein to lipid vesicles with high negative charge content is essentially unaffected by N-terminal acetylation irrespective of curvature, but binding to vesicles of lower negative charge content is increased, with stronger binding observed for vesicles with higher curvature. Thus, the naturally occurring N-terminally acetylated form of α-synuclein exhibits stabilized helicity at its N terminus and increased affinity for lipid vesicles similar to synaptic vesicles, a binding target of the protein in vivo. Furthermore, the novel BOG-bound state of N-terminally acetylated α-synuclein may serve as a model of partly helical membrane-bound intermediates with a role in α-synuclein function and dysfunction.