RESUMEN
Although ovalbumin (OVA), a main component of hen egg white and a non-inhibitory serpin superfamily protein, has been reported to form fibrillar aggregates, its relationship with amyloid fibrils associated with various degenerative diseases is unclear. We studied the heat-induced aggregation of intact OVA using an amyloid-specific thioflavin T assay with a fluorometer or direct imaging with a light-emitting diode lamp and several physicochemical approaches, and the results confirmed that intact OVA forms aggregates with a small part of amyloid cores and dominantly amorphous aggregates. We isolated the amyloidogenic core peptide by proteolysis with trypsin. The isolated 23-residue peptide, pOVA, with marked amyloidogenicity, corresponded to one (ß-strand 3A) of the key regions involved in serpin latency transition and domain-swap polymerization leading to serpinopathies. Although the strong amyloidogenicity of pOVA was suppressed in a mixture of tryptic digests, it was observed under acidic conditions in the presence of various salts, with which pOVA has a positive charge. Cytotoxicity measurements suggested that, although heat-treated OVA aggregates exhibited the strongest toxicity, it was attributed to a general property of amorphous aggregates rather than amyloid toxicity. Predictions indicated that the high amyloidogenicity of the ß-strand 3A region is common to various serpins. This suggests that the high amyloidogenicity of ß-strand 3A that is important for serpin latency transition and domain-swap polymerization is retained in OVA and constitutes ß-spine amyloid cores upon heat aggregation.
Asunto(s)
Amiloide/farmacología , Neoplasias del Colon/patología , Calor , Ovalbúmina/química , Agregado de Proteínas , Serpinas/química , Amiloide/química , Animales , Pollos , Neoplasias del Colon/tratamiento farmacológico , Ratones , Polimerizacion , Células Tumorales CultivadasRESUMEN
Synaptic plasticity is long-lasting changes in synaptic currents and structure. When neurons are exposed to signals that induce aberrant neuronal excitation, they increase the threshold for the induction of long-term potentiation (LTP), known as metaplasticity. However, the metaplastic regulation of structural LTP (sLTP) remains unclear. We investigate glutamate uncaging/photoactivatable (pa)CaMKII-dependent sLTP induction in hippocampal CA1 neurons after chronic neuronal excitation by GABAA receptor antagonists. We find that the neuronal excitation decreases the glutamate uncaging-evoked Ca2+ influx mediated by GluN2B-containing NMDA receptors and suppresses sLTP induction. In addition, single-spine optogenetic stimulation using paCaMKII indicates the suppression of CaMKII signaling. While the inhibition of Ca2+ influx is protein synthesis independent, the paCaMKII-induced sLTP suppression depends on it. Our findings demonstrate that chronic neuronal excitation suppresses sLTP in two independent ways (i.e., dual inhibition of Ca2+ influx and CaMKII signaling). This dual inhibition mechanism may contribute to robust neuronal protection in excitable environments.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Potenciación a Largo Plazo/fisiología , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/fisiología , Animales , Región CA1 Hipocampal/metabolismo , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Línea Celular , Espinas Dendríticas/metabolismo , Antagonistas de Receptores de GABA-A/farmacología , Ácido Glutámico/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Receptores de GABA-A/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Optogenetic approaches for studying neuronal functions have proven their utility in the neurosciences. However, optogenetic tools capable of inducing synaptic plasticity at the level of single synapses have been lacking. Here, we engineered a photoactivatable (pa)CaMKII by fusing a light-sensitive domain, LOV2, to CaMKIIα. Blue light or two-photon excitation reversibly activated paCaMKII. Activation in single spines was sufficient to induce structural long-term potentiation (sLTP) in vitro and in vivo. paCaMKII activation was also sufficient for the recruitment of AMPA receptors and functional LTP in single spines. By combining paCaMKII with protein activity imaging by 2-photon FLIM-FRET, we demonstrate that paCaMKII activation in clustered spines induces robust sLTP via a mechanism that involves the actin-regulatory small GTPase, Cdc42. This optogenetic tool for dissecting the function of CaMKII activation (i.e., the sufficiency of CaMKII rather than necessity) and for manipulating synaptic plasticity will find many applications in neuroscience and other fields.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Potenciación a Largo Plazo/fisiología , Optogenética/métodos , Sinapsis/metabolismo , Animales , Electrofisiología , Femenino , Células HeLa , Hipocampo/metabolismo , Hipocampo/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Plasticidad Neuronal/fisiología , Receptores AMPA/genética , Receptores AMPA/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiología , Sinapsis/fisiologíaRESUMEN
Here we developed an orange light-absorbing chromoprotein named ShadowR as a novel acceptor for performing fluorescence lifetime imaging microscopy-based Förster resonance energy transfer (FLIM-FRET) measurement in living cells. ShadowR was generated by replacing hydrophobic amino acids located at the surface of the chromoprotein Ultramarine with hydrophilic amino acids in order to reduce non-specific interactions with cytosolic proteins. Similar to Ultramarine, ShadowR shows high absorption capacity and no fluorescence. However, it exhibits reduced non-specific binding to cytosolic proteins and is highly expressed in HeLa cells. Using tandem constructs and a LOVTRAP system, we showed that ShadowR can be used as a FRET acceptor in combination with donor mRuby2 or mScarlet in HeLa cells. Thus, ShadowR is a useful, novel FLIM-FRET acceptor.
Asunto(s)
Fenómenos Biofísicos , Fluorescencia , Proteínas Luminiscentes/química , Microscopía Fluorescente/métodos , Transferencia Resonante de Energía de Fluorescencia , Expresión Génica/genética , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Unión Proteica/genéticaRESUMEN
Complex formation of gramicidin (GA) and desformylgramicidin (des-GA) with sterols was investigated by measuring the intrinsic Trp fluorescence. In organic solvents, the Trp fluorescence of momeric GA was quenched upon binding either cholesterol or ergosterol, but that of monomeric des-GA was not quenched by adding cholesterol. Both dimeric GA and des-GA bound highly to ergosterol, but not to cholesterol, determined by quenching of Trp fluorescence. Furthermore, GA- and des-GA-loaded lysophosphatidylcholine micelles were incubated with phosphatidylcholine vesicles containing cholesterol or ergosterol. The results showed that both monomeric and dimeric peptides hardly bound to cholesterol incorporated into phospholipid vesicles, but markedly bound to ergosterol incorporated into the bilayer membranes. Interestingly, des-GA bound more specifically to the two sterols than GA. In addition, fluorescence resonance energy transfer analysis showed that des-GA bound more specifically to the two sterol than GA.
Asunto(s)
Antibacterianos/química , Colesterol/metabolismo , Ergosterol/metabolismo , Gramicidina/análogos & derivados , Gramicidina/química , Fosfolípidos/química , Antibacterianos/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Gramicidina/metabolismo , Lisofosfatidilcolinas/química , Micelas , Fosfatidilcolinas/química , Fosfolípidos/metabolismo , Solventes/químicaRESUMEN
We review here the molecular mechanisms that underlie alpha1-antitrypsin deficiency and show how an understanding of this mechanism has allowed us to explain the deficiency of other members of the serine proteinase inhibitor or serpin superfamily. These include the deficiency of antithrombin, C1-inhibitor and alpha1-antichymotrypsin in association with thrombosis, angio-oedema and emphysema respectively. Moreover the accumulation of mutant neuroserpin within neurones causes the novel dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB). We have grouped these conditions together as the serpinopathies as recognition of their common pathophysiology provides a platform to develop strategies to treat the associated clinical syndromes.
Asunto(s)
Demencia/patología , Deficiencia de alfa 1-Antitripsina/patología , alfa 1-Antitripsina/química , Alelos , Animales , Antitrombinas/química , Antitrombinas/metabolismo , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Enfisema/patología , Humanos , Hígado/metabolismo , Enfermedades Pulmonares/patología , Mutación , Neuropéptidos/química , Inhibidor 1 de Activador Plasminogénico/química , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 2 de Activador Plasminogénico/química , Inhibidor 2 de Activador Plasminogénico/genética , Polímeros/química , Estructura Terciaria de Proteína , Serpinas/química , Trombosis/patología , alfa 1-Antitripsina/genética , NeuroserpinaRESUMEN
To investigate the susceptibilities of fungal and mammalian cells to amphotericin B (AmB), AmB-loaded lysophosphatidylcholine (LPC)micelles as drug delivery vehicles were incubated at 37 degrees C with phosphatidylcholine vesicles containing different sterols as model systems for fungal and mammalian cells. The binding and kinetics of AmB to sterols in the membranes were judged by UV-visible spectroscopy. In the 91% monomeric form, AmB interacted rapidly with ergosterol and slowly with 7-dehydrocholesterol (7-DHC), while it did not interact with cholesterol. In the 50% monomeric form, AmB formed complexes more rapidly with ergosterol or 7-DHC than in the monomeric form, whereas it did not still interact with cholesterol. The interaction was also characterized by resonance energy transfer between the fluorescent probe trimethylammonium diphenylhexatriene (TMA-DPH) and AmB. In the 91% monomeric form, AmB caused initial fluorescence quenching in bilayer membranes containing any sterol as well as sterol-free bilayer membranes due to the release of AmB and its incorporation within the membranes. However, a second phase of increasing fluorescence was found in the case of ergosterol alone. On the other hand, in the 47% monomeric form, AmB gave a biphasic intensity profile in membranes containing any sterol as well as sterol-free membranes. However, the extent of the second phase of increasing fluorescence intensity was markedly dependent upon sterol composition. Studies using sterol-containing vesicles provide important insights into the role of the aggregation state of AmB in its effects on cells.
Asunto(s)
Anfotericina B/química , Dimiristoilfosfatidilcolina/química , Liposomas/química , Lisofosfatidilcolinas/química , Micelas , Esteroles/química , Colesterol/química , Deshidrocolesteroles/química , Portadores de Fármacos/química , Ergosterol/química , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
Neuroserpin is a member of the serpin superfamily. Point mutations in the neuroserpin gene underlie the autosomal dominant dementia, familial encephalopathy with neuroserpin inclusion bodies. This is characterized by the retention of ordered polymers of neuroserpin within the endoplasmic reticulum of neurons. pH has been shown to affect the propensity of several serpins to form polymers. In particular, low pH favors the formation of polymers of both alpha(1)-antitrypsin and antithrombin. We report here opposite effects in neuroserpin, with a striking resistance to polymer formation at acidic pH. Mutation of specific histidine residues showed that this effect is not attributable to the shutter domain histidine as would be predicted by analogy with other serpins. Indeed, mutation of the shutter domain His338 decreased neuroserpin stability but had no effect on the pH dependence of polymerization when compared with the wild-type protein. In contrast, mutation of His119 or His138 reduced the polymerization of neuroserpin at both acidic and neutral pH. These residues are at the lower pole of neuroserpin and provide a novel mechanism to control the opening of beta-sheet A and hence polymerization. This mechanism is likely to have evolved to protect neuroserpin from the acidic environment of the secretory granules.
Asunto(s)
Neuropéptidos/química , Neuropéptidos/metabolismo , Serpinas/química , Serpinas/metabolismo , Histidina/genética , Histidina/metabolismo , Concentración de Iones de Hidrógeno , Modelos Moleculares , Mutación , Neuropéptidos/genética , Unión Proteica , Estabilidad Proteica , Estructura Secundaria de Proteína , Serpinas/genética , NeuroserpinaRESUMEN
Neuroserpin is a member of the serpin superfamily, and its mutants are retained within the endoplasmic reticulum of neurons as ordered polymers in association with dementia. It has been proposed that neuroserpin polymers are formed by a conformational change in the folded protein. However, an alternative model whereby polymers are formed during protein folding rather than from the folded protein has recently been proposed. We investigated the refolding and polymerization pathways of wild-type neuroserpin (WT) and of the pathogenic mutants S49P and H338R. Upon refolding, denatured WT immediately formed an initial refolding intermediate I(IN) and then underwent further refolding to the native form through a late refolding intermediate, I(R). The late-onset mutant S49P was also able to refold to the native form through I(IN) and I(R), but the final refolding step proceeded at a slower rate and with a lower refolding yield as compared with WT. The early-onset mutant H338R formed I(R) through the same pathway as S49P, but the protein could not attain the native state and remained as I(R). The I(R)s of the mutants had a long lifespan at 4 °C and thus were purified and characterized. Strikingly, when incubated under physiological conditions, I(R) formed ordered polymers with essentially the same properties as the polymers formed from the native protein. The results show that the mutants have a greater tendency to form polymers during protein folding than to form polymers from the folded protein. Our finding provides insights into biochemical approaches to treating serpinopathies by targeting a polymerogenic folding intermediate.
Asunto(s)
Neuropéptidos/química , Serpinas/química , Sustitución de Aminoácidos , Encéfalo/metabolismo , Dicroismo Circular , Demencia/genética , Demencia/metabolismo , Humanos , Técnicas In Vitro , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neuropéptidos/genética , Neuropéptidos/metabolismo , Pliegue de Proteína , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serpinas/genética , Serpinas/metabolismo , Espectrometría de Fluorescencia , NeuroserpinaRESUMEN
Neuroserpin is a selective inhibitor of tissue-type plasminogen activator (tPA) that plays an important role in neuronal plasticity, memory, and learning. We report here the crystal structure of native human neuroserpin at 2.1 A resolution. The structure has a helical reactive center loop and an omega loop between strands 1B and 2B. The omega loop contributes to the inhibition of tPA, as deletion of this motif reduced the association rate constant with tPA by threefold but had no effect on the kinetics of interaction with urokinase. Point mutations in neuroserpin cause the formation of ordered intracellular polymers that underlie dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type neuroserpin is also unstable and readily forms polymers under near-physiological conditions in vitro. This is, in part, due to the substitution of a conserved alanine for serine at position 340. The replacement of Ser340 by Ala increased the melting temperature by 3 degrees C and reduced polymerization as compared to wild-type neuroserpin. Similarly, neuroserpin has Asn-Leu-Val at the end of helix F and thus differs markedly from the Gly-X-Ile consensus sequence of the serpins. Restoration of these amino acids to the consensus sequence increased thermal stability and reduced the polymerization of neuroserpin and its transition to the latent conformer. Moreover, introduction of the consensus sequence into S49P neuroserpin that causes FENIB increased the stability and inhibitory activity of the mutant, as well as blocked polymerization and increased the yield of protein during refolding. These data provide a molecular explanation for the inherent instability of neuroserpin and the effect of point mutations that underlie the dementia FENIB.
Asunto(s)
Neuropéptidos/química , Serpinas/química , Secuencia Conservada , Cristalografía por Rayos X , Demencia/metabolismo , Humanos , Cuerpos de Inclusión/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Neuropéptidos/metabolismo , Mutación Puntual , Conformación Proteica , Pliegue de Proteína , Alineación de Secuencia , Serpinas/metabolismo , Activador de Tejido Plasminógeno/metabolismo , NeuroserpinaRESUMEN
Serine proteinase inhibitors (serpins) are believed to fold in vivo into a metastable "stressed" state with cleavage of their P1-P1' bond resulting in reactive center loop insertion and a thermostable "relaxed" state. To understand this unique folding mechanism, we investigated the refolding processes of the P1-P1'-cleaved forms of wild type ovalbumin (cl-OVA) and the R339T mutant (cl-R339T). In the native conditions, cl-OVA is trapped as the stressed conformer, whereas cl-R339T attains the relaxed structure. Under urea denaturing conditions, these cleaved proteins completely dissociated into the heavy (Gly(1)-Ala(352)) and light (Ser(353)-Pro(385)) chains. Upon refolding, the heavy chains of both proteins formed essentially the same initial burst refolding intermediates and then reassociated with the light chain counterparts. The reassociated intermediates both refolded into the native states with indistinguishable kinetics. The two refolded proteins, however, had a notable difference in thermostability. cl-OVA refolded into the stressed form with T(m) = 68.4 degrees C, whereas cl-R339T refolded into the relaxed form with T(m) = 85.5 degrees C. To determine whether cl-R339T refolds directly to the relaxed state or through the stressed state, conformational analyses by anion-exchange chromatography and fluorescence measurements were executed. The results showed that cl-R339T refolds first to the stressed conformation and then undergoes the loop insertion. This is the first demonstration that the P1-P1'-cleaved serpin peptide capable of loop insertion refolds to the stressed conformation. This highlights that the stressed conformation of serpins is an inevitable intermediate state on the folding pathway to the relaxed structure.
Asunto(s)
Serpinas/química , Animales , Aniones , Rastreo Diferencial de Calorimetría/métodos , Pollos , Cromatografía por Intercambio Iónico/métodos , Dicroismo Circular , Clara de Huevo , Cinética , Modelos Químicos , Conformación Molecular , Unión Proteica , Conformación Proteica , Desnaturalización Proteica , Pliegue de ProteínaRESUMEN
The serpinopathies result from conformational transitions in members of the serine proteinase inhibitor superfamily with aberrant tissue deposition or loss of function. They are typified by mutants of neuroserpin that are retained within the endoplasmic reticulum of neurons as ordered polymers in association with dementia. We show here that the S49P mutant of neuroserpin that causes the dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) forms a latent species in vitro and in vivo in addition to the formation of polymers. Latent neuroserpin is thermostable and inactive as a proteinase inhibitor, but activity can be restored by refolding. Strikingly, latent S49P neuroserpin is unlike any other latent serine proteinase inhibitor (serpin) in that it spontaneously forms polymers under physiological conditions. These data provide an alternative method for the inactivation of mutant neuroserpin as a proteinase inhibitor in FENIB and demonstrate a second pathway for the formation of intracellular polymers in association with disease.
Asunto(s)
Biopolímeros/química , Demencia/genética , Demencia/patología , Cuerpos de Inclusión/química , Neuropéptidos , Mutación Puntual , Serpinas , Adulto , Animales , Biopolímeros/metabolismo , Células COS , Chlorocebus aethiops , Ácido Cítrico/química , Demencia/metabolismo , Humanos , Persona de Mediana Edad , Modelos Moleculares , Neuronas/citología , Neuronas/metabolismo , Neuropéptidos/química , Neuropéptidos/genética , Neuropéptidos/aislamiento & purificación , Neuropéptidos/metabolismo , Conformación Proteica , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serpinas/química , Serpinas/genética , Serpinas/aislamiento & purificación , Serpinas/metabolismo , Temperatura , NeuroserpinaRESUMEN
Ovalbumin, a member of the serpin superfamily, is transformed into a thermostabilized form, S-ovalbumin, during storage of shell eggs or by an alkaline treatment of the isolated protein (DeltaT(m)=8 degrees C). As structural characteristics of S-ovalbumin, three serine residues (Ser164, Ser236 and Ser320) take the D-amino acid residue configuration, while the conformational change from non-thermostabilized native ovalbumin is very small. To assess the role of the structural characteristics on protein thermostabilization, ovalbumin and S-ovalbumin were denatured to eliminate the conformational modulation effects and then refolded. The denatured ovalbumin and S-ovalbumin were correctly refolded into the original non-denatured forms with the corresponding differential thermostability. There was essentially no difference in the disulfide structures of the native and refolded forms of ovalbumin and S-ovalbumin. These data are consistent with the view that the configuration inversion, which is the only chemical modification directly detected in S-ovalbumin so far, plays a central role in ovalbumin thermostabilization. The rate of refolding of S-ovalbumin was greater than that of ovalbumin, indicating the participation, at least in part, of an increased folding rate for thermodynamic stabilization.
Asunto(s)
Calor , Ovalbúmina/química , Pliegue de Proteína , Cinética , Desnaturalización Proteica , Renaturación de Proteína , Factores de TiempoRESUMEN
Ovalbumin, a member of the serpin superfamily, contains one cystine disulfide (Cys73-Cys120) and four cysteine sulfhydryls (Cys11, Cys30, Cys367, and Cys382) in the native state. To investigate the folding mechanism of ovalbumin, a urea-denatured disulfide isomer with a mispaired disulfide Cys367-Cys382 (D[367-382]) and its derivative (D[367-382/CM-73]) in which a native cystine counterpart of Cys73 is blocked by carboxymethylation were produced. Both the denatured isomers refolded within an instrumental dead time of 4 ms into an initial burst intermediate IN with partially folded conformation. After the initial burst phase, most of the D[367-382] molecules further refolded into the native form. In contrast, upon dilution of D[367-382/CM-73] with the refolding buffer, the protein stayed in the IN state as a stable form, which displayed a partial regain of the native secondary structure and a compact conformation with a similar Stokes radius to the native form. The structural characteristics of IN were clearly differentiated from those of an equilibrium intermediate IA that was produced by dilution with an acidic buffer of urea-denatured ovalbumin; IA showed much more hydrophobic dye binding and a larger Stokes radius than the IN state, despite their indistinguishable far-UV circular dichroic spectra. The non-productive nature of IA highlighted the importance of a compact conformation of the IN state for subsequent native refolding. These observations were consistent with a refolding model of ovalbumin that includes the regain of the partial secondary structure and of the compactness of overall conformation in an initial burst phase before the subsequent native refolding.
Asunto(s)
Ovalbúmina/química , Renaturación de Proteína , Animales , Rastreo Diferencial de Calorimetría , Pollos , Cistina , Isomerismo , Cinética , Mapeo Peptídico , Conformación Proteica , Pliegue de Proteína , Urea/farmacologíaRESUMEN
The dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) is caused by point mutations in the neuroserpin gene. We have shown a correlation between the predicted effect of the mutation and the number of intracerebral inclusions, and an inverse relationship with the age of onset of disease. Our previous work has shown that the intraneuronal inclusions in FENIB result from the sequential interaction between the reactive centre loop of one neuroserpin molecule with beta-sheet A of the next. We show here that neuroserpin Portland (Ser52Arg), which causes a severe form of FENIB, also forms loop-sheet polymers but at a faster rate, in keeping with the more severe clinical phenotype. The Portland mutant has a normal unfolding transition in urea and a normal melting temperature but is inactive as a proteinase inhibitor. This results in part from the reactive loop being in a less accessible conformation to bind to the target enzyme, tissue plasminogen activator. These results, with those of the CD analysis, are in keeping with the reactive centre loop of neuroserpin Portland being partially inserted into beta-sheet A to adopt a conformation similar to an intermediate on the polymerization pathway. Our data provide an explanation for the number of inclusions and the severity of dementia in FENIB associated with neuroserpin Portland. Moreover the inactivity of the mutant may result in uncontrolled activity of tissue plasminogen activator, and so explain the epileptic seizures seen in individuals with more severe forms of the disease.