RESUMEN
Free fatty acids (FFA) cause apoptosis of pancreatic beta-cells and might contribute to beta-cell loss in type 2 diabetes via the induction of endoplasmic reticulum (ER) stress. We studied here the molecular mechanisms implicated in FFA-induced ER stress initiation and apoptosis in INS-1E cells, FACS-purified primary beta-cells and human islets exposed to oleate and/or palmitate. Treatment with saturated and/or unsaturated FFA led to differential ER stress signaling. Palmitate induced more apoptosis and markedly activated the IRE1, PERK and ATF6 pathways, owing to a sustained depletion of ER Ca(2+) stores, whereas the unsaturated FFA oleate led to milder PERK and IRE1 activation and comparable ATF6 signaling. Non-metabolizable methyl-FFA analogs induced neither ER stress nor beta-cell apoptosis. The FFA-induced ER stress response was not modified by high glucose concentrations, suggesting that ER stress in primary beta-cells is primarily lipotoxic, and not glucolipotoxic. Palmitate, but not oleate, activated JNK. JNK inhibitors reduced palmitate-mediated AP-1 activation and apoptosis. Blocking the transcription factor CHOP delayed palmitate-induced beta-cell apoptosis. In conclusion, saturated FFA induce ER stress via ER Ca(2+) depletion. The IRE1 and resulting JNK activation contribute to beta-cell apoptosis. PERK activation by palmitate also contributes to beta-cell apoptosis via CHOP.
Asunto(s)
Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/patología , Ácidos Grasos no Esterificados/toxicidad , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/patología , Factor de Transcripción Activador 3/metabolismo , Factor de Transcripción Activador 6/metabolismo , Animales , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Retículo Endoplásmico/enzimología , Ácidos Grasos no Esterificados/metabolismo , Glucosa/toxicidad , Humanos , Células Secretoras de Insulina/enzimología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Persona de Mediana Edad , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Factor de Transcripción CHOP/metabolismo , eIF-2 Quinasa/metabolismoRESUMEN
In native reaction centers (RCs) from photosynthetic purple bacteria the primary quinone (QA) and the secondary quinone (QB) are interconnected via a specific His-Fe-His bridge. In Rhodobacter sphaeroides RCs the C4=O carbonyl of QA forms a very strong hydrogen bond with the protonated Npi of His M219, and the Ntau of this residue is in turn coordinated to the non-heme iron atom. The second carbonyl of QA is engaged in a much weaker hydrogen bond with the backbone N-H of Ala M260. In previous work, a Trp side chain was introduced by site-directed mutagenesis at the M260 position in the RC of Rb. sphaeroides, resulting in a complex that is completely devoid of QA and therefore nonfunctional. A photochemically competent derivative of the AM260W mutant was isolated that contains a Cys side chain at the M260 position (denoted AM260(W-->C)). In the present work, the interactions between the carbonyl groups of QA and the protein in the AM260(W-->C) suppressor mutant have been characterized by light-induced FTIR difference spectroscopy of the photoreduction of QA. The QA-/QA difference spectrum demonstrates that the strong interaction between the C4=O carbonyl of QA and His M219 is lost in the mutant, and the coupled CO and CC modes of the QA- semiquinone are also strongly perturbed. In parallel, a band assigned to the perturbation of the C5-Ntau mode of His M219 upon QA- formation in the native RC is lacking in the spectrum of the mutant. Furthermore, a positive band between 2900 and 2400 cm-1 that is related to protons fluctuating within a network of highly polarizable hydrogen bonds in the native RC is reduced in amplitude in the mutant. On the other hand, the QB-/QB FTIR difference spectrum is essentially the same as for the native RC. The kinetics of electron transfer from QA- to QB were measured by the flash-induced absorption changes at 780 nm. Compared to native RCs the absorption transients are slowed by a factor of about 2 for both the slow phase (in the hundreds of microseconds range) and fast phase (microseconds to tens of microseconds range) in AM260(W-->C) RCs. We conclude that the unusually strong hydrogen bond between the carbonyl of QA and His M219 in the Rb. sphaeroides RC is not obligatory for efficient electron transfer from QA- to QB.
Asunto(s)
Histidina/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Quinonas/metabolismo , Rhodobacter sphaeroides/metabolismo , Sitios de Unión/genética , Cisteína/química , Cisteína/genética , Transporte de Electrón/genética , Histidina/química , Histidina/genética , Enlace de Hidrógeno , Hierro/metabolismo , Cinética , Luz , Modelos Moleculares , Estructura Molecular , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Conformación Proteica , Rhodobacter sphaeroides/genética , Espectroscopía Infrarroja por Transformada de Fourier , AguaRESUMEN
The purple photosynthetic bacterium Thermochromatium tepidum is a moderate thermophile, with a growth optimum of 48-50 degrees C. The X-ray crystal structure of the reaction centre from this organism has been determined, and compared with that from mesophilic bacteria such as Blastochloris viridis and Rhodobacter sphaeroides (Nogi T et al. (2000) Proc Natl Acad Sci USA 97: 13561-13566). Structural features that could contribute to the enhanced thermal stability of the Thermochromatium tepidum reaction centre were discussed, including three arginine residues exposed at the periplasmic side of the membrane that are not present in reaction centres from mesophilic organisms, and potentially could increase the affinity of the complex for the surrounding membrane. In the present report these arginine residues, plus a histidine identified from an extensive sequence alignment, were engineered into structurally homologous positions in the Rhodobacter sphaeroides reaction centre, and the effect on the thermal stability of the Rhodobacter sphaeroides complex was examined. We find that these residues do not enhance the thermal stability of the reaction centre, as assessed by absorbance spectroscopy of the bacteriochlorin cofactors in membrane-bound reaction centres. Possible roles of these residues in the Thermochromatium tepidum reaction centre are discussed, and it is proposed that they facilitate stronger binding of the reaction centre to the encircling LH1 antenna complex, through ionic interactions with acidic residues at the C-terminal end of the LH1 alpha-polypeptide. Such an interaction could enhance the stability of the so-called 'RC-LH1 core' complex that is formed between the reaction centre and the LH1 antenna, and which represents the minimal functional photosynthetic unit in all known purple photosynthetic bacteria. Stronger bonding interactions between the two complexes could also contribute to an increase in the rigidity of the photosynthetic membrane in Thermochromatium tepidum, in accord with the general finding that the cytoplasmic membrane from thermophilic eubacteria is less fluid than its counterpart in mesophilic bacteria.
Asunto(s)
Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Proteobacteria/crecimiento & desarrollo , Proteobacteria/metabolismo , Temperatura , Proliferación Celular , Estabilidad de Enzimas , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Mutación/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Unión Proteica , Desnaturalización Proteica , Estructura Terciaria de Proteína , Proteobacteria/genética , Espectroscopía Infrarroja CortaRESUMEN
All of the membrane-embedded cofactors of the purple bacterial reaction centre have well-defined functional or structural roles, with the exception of the bacteriopheophytin (H(B)) located approximately half-way across the membrane on the so-called inactive- or B-branch of cofactors. Sequence alignments indicate that this bacteriochlorin cofactor is a conserved feature of purple bacterial reaction centres, and a pheophytin is also found at this position in the Photosystem-II reaction centre. Possible structural or functional consequences of replacing the H(B) bacteriopheophytin by bacteriochlorophyll were investigated in the Rhodobacter sphaeroides reaction centre through mutagenesis of residue Leu L185 to His (LL185H). Results from absorbance spectroscopy indicated that the LL185H mutant assembled with a bacteriochlorophyll at the H(B) position, but this did not affect the capacity of the reaction centre to support photosynthetic growth, or change the kinetics of charge separation along the A-branch of cofactors. It was also found that mutation of residue Ala M149 to Trp (AM149W) caused the reaction centre to assemble without an H(B) bacteriochlorin, demonstrating that this cofactor is not required for correct assembly of the reaction centre. The absence of a cofactor at this position did not affect the capacity of the reaction centre to support photosynthetic growth, or the kinetics of A-branch electron transfer. A combination of X-ray crystallography and FTIR difference spectroscopy confirmed that the H(B) cofactor was absent in the AM149W mutant, and that this had not produced any significant disturbance of the adjacent ubiquinol reductase (Q(B)) site. The data are discussed with respect to possible functional roles of the H(B) bacteriopheophytin, and we conclude that the reason(s) for conservation of a bacteriopheophytin cofactor at this position in purple bacterial reaction centres are likely to be different from those underlying conservation of a pheophytin at the analogous position in Photosystem-II.
Asunto(s)
Feofitinas/química , Feofitinas/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Rhodobacter sphaeroides/química , Rhodobacter sphaeroides/metabolismo , Clorofila , Color , Cristalografía por Rayos X , Modelos Moleculares , Conformación Molecular , Mutación/genética , Fenotipo , Feofitinas/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Rhodobacter sphaeroides/genética , Análisis Espectral , TemperaturaRESUMEN
The dynamics of electron transfer in a membrane-bound Rhodobacter sphaeroides reaction centre containing a combination of four mutations were investigated by transient absorption spectroscopy. The reaction centre, named WAAH, has a mutation that causes the reaction centre to assemble without a Q(A) ubiquinone (Ala M260 to Trp), a mutation that causes the replacement of the H(A) bacteriopheophytin with a bacteriochlorophyll (Leu M214 to His) and two mutations that remove acidic groups close to the Q(B) ubiquinone (Glu L212 to Ala and Asp L213 to Ala). Previous work has shown that the Q(B) ubiquinone is reduced by electron transfer along the so-called inactive cofactor branch (B-branch) in the WAAH reaction centre (M.C. Wakeham, M.G. Goodwin, C. McKibbin, M.R. Jones, Photo-accumulation of the P(+)Q(B)(-) radical pair state in purple bacterial reaction centres that lack the Q(A) ubiquinone, FEBS Letters 540 (2003) 234-240). In the present study the dynamics of electron transfer in the membrane-bound WAAH reaction centre were studied by femtosecond transient absorption spectroscopy, and the data analysed using a compartmental model. The analysis indicates that the yield of Q(B) reduction via the B-branch is approximately 8% in the WAAH reaction centre, consistent with results from millisecond time-scale kinetic spectroscopy. Possible contributions to this yield of the constituent mutations in the WAAH reaction centre and the membrane environment of the complex are discussed.
Asunto(s)
Transporte de Electrón , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Rhodobacter sphaeroides/química , Espectrofotometría/métodos , Análisis de Fourier , Cinética , Modelos Químicos , Mutagénesis Sitio-Dirigida , Mutación Missense , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Factores de Tiempo , Ubiquinona/deficienciaRESUMEN
The role of a water molecule (water A) located between the primary electron donor (P) and first electron acceptor bacteriochlorophyll (B(A)) in the purple bacterial reaction center was investigated by mutation of glycine M203 to leucine (GM203L). The x-ray crystal structure of the GM203L reaction center shows that the new leucine residue packs in such a way that water A is sterically excluded from the complex, but the structure of the protein-cofactor system around the mutation site is largely undisturbed. The results of absorbance and resonance Raman spectroscopy were consistent with either the removal of a hydrogen bond interaction between water A and the keto carbonyl group of B(A) or a change in the local electrostatic environment of this carbonyl group. Similarities in the spectroscopic properties and x-ray crystal structures of reaction centers with leucine and aspartic acid mutations at the M203 position suggested that the effects of a glycine to aspartic acid substitution at the M203 position can also be explained by steric exclusion of water A. In the GM203L mutant, loss of water A was accompanied by an approximately 8-fold slowing of the rate of decay of the primary donor excited state, indicating that the presence of water A is important for optimization of the rate of primary electron transfer. Possible functions of this water molecule are discussed, including a switching role in which the redox potential of the B(A) acceptor is rapidly modulated in response to oxidation of the primary electron donor.
Asunto(s)
Bacterioclorofilas/química , Transporte de Electrón , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Rhodobacter sphaeroides/química , Agua/química , Sustitución de Aminoácidos , Cristalografía por Rayos X , Transporte de Electrón/genética , Transporte de Electrón/efectos de la radiación , Enlace de Hidrógeno , Cinética , Luz , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Análisis EspectralRESUMEN
In Rhodobacter sphaeroides reaction centers (RCs) containing the mutation Ala M260 to Trp (AM260W), transmembrane electron transfer along the full-length of the A-branch of cofactors is prevented by the loss of the Q(A) ubiquinone, but it is possible to generate the radical pair P(+)H(A)(-) by A-branch electron transfer or the radical pair P(+)Q(B)(-) by B-branch electron transfer. In the present study, FTIR spectroscopy was used to provide direct evidence for the complete absence of the Q(A) ubiquinone in mutant RCs with the AM260W mutation. Light-induced FTIR difference spectroscopy of isolated RCs was also used to probe the neutral Q(B) and the semiquinone Q(B)(-) states in two B-branch active mutants, a double AM260W-LM214H mutant, denoted WH, and a quadruple mutant, denoted WAAH, in which the AM260W, LM214H, and EL212A-DL213A mutations were combined. The data were compared to those obtained with wild-type (Wt) RCs and the double EL212A-DL213A (denoted AA) mutant which exhibit the usual A-branch electron transfer to Q(B). The Q(B)(-)/Q(B) spectrum of the WH mutant is very close to that of Wt RCs indicating similar bonding interactions of Q(B) and Q(B)(-) with the protein in both RCs. The Q(B)(-)/Q(B) spectra of the AA and WAAH mutants are also closely related to one another, but are very different to that of the Wt complex. Isotope-edited IR fingerprint spectra were obtained for the AA and WAAH mutants reconstituted with site-specific (13)C-labeled ubiquinone. Whilst perturbations of the interactions of the semiquinone Q(B)(-) with the protein are observed in the AA and WAAH mutants, the FTIR data show that the bonding interaction of neutral Q(B) in these two mutants are essentially the same as those for Wt RCs. Therefore, it is concluded that Q(B) occupies the same binding position proximal to the non-heme iron prior to reduction by either A-branch or B-branch electron transfer.
Asunto(s)
Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismo , Ubiquinona/metabolismo , Sustitución de Aminoácidos , Sitios de Unión/genética , Isótopos de Carbono/metabolismo , Transporte de Electrón , Modelos Moleculares , Estructura Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fotoquímica , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Rhodobacter sphaeroides/química , Espectroscopía Infrarroja por Transformada de Fourier , Triptófano/metabolismo , Ubiquinona/químicaRESUMEN
In Rhodobacter sphaeroides reaction centers containing the mutation Ala M260 to Trp (AM260W), transmembrane electron transfer along the A-branch of cofactors is prevented by the loss of the QA ubiquinone. Reaction centers that contain this AM260W mutation are proposed to photoaccumulate the P(+)QB- radical pair following transmembrane electron transfer along the B-branch of cofactors (Wakeham, M. C., Goodwin, M. G., McKibbin, C., and Jones, M. R. (2003) Photoaccumulation of the P(+)QB- radical pair state in purple bacterial reaction centers that lack the QA ubiquinone. FEBS Lett. 540, 234-240). The yield of the P(+)QB- state appears to depend upon which additional mutations are present. In the present paper, Fourier transform infrared (FTIR) difference spectroscopy was used to demonstrate that photooxidation of the reaction center's primary donor in QA-deficient reaction centers results in formation of a semiquinone at the QB site by B-branch electron transfer. Reduction of QB by the B-branch pathway still occurs at 100 K, with a yield of approximately 10% relative to that at room temperature, in contrast to the QA- to QB reaction in the wild-type reaction center, which is not active at cryogenic temperatures. These FTIR results suggest that the conformational changes that "gate" the QA- to QB reaction do not necessarily have the same influence on QB reduction when the electron donor is the HB anion, at least in a minority of reaction centers.
Asunto(s)
Benzoquinonas/química , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Rhodobacter sphaeroides/química , Alanina/genética , Antibacterianos/química , Benzoquinonas/antagonistas & inhibidores , Benzoquinonas/metabolismo , Sitios de Unión/genética , Transporte de Electrón/genética , Congelación , Luz , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Proteínas del Complejo del Centro de Reacción Fotosintética/antagonistas & inhibidores , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Polienos/química , Rhodobacter sphaeroides/genética , Espectroscopía Infrarroja por Transformada de Fourier , Triptófano/genética , Ubiquinona/genética , Ubiquinona/metabolismoRESUMEN
A photosynthetically impaired strain of Rhodobacter sphaeroides containing reaction centres with an alanine to tryptophan mutation at residue 260 of the M-polypeptide (AM260W) was incubated under photosynthetic growth conditions. This incubation produced photosynthetically competent strains containing suppressor mutations that changed residue M260 to glycine or cysteine. Spectroscopic analysis demonstrated that the loss of the Q(A) ubiquinone seen in the original AM260W mutant was reversed in the suppressor mutants. In the mutant where Trp M260 was replaced by Cys, the rate of reduction of the Q(A) ubiquinone by the adjacent (H(A)) bacteriopheophytin was reduced by three-fold. The findings of the experiment are discussed in light of the X-ray crystal structures of the wild-type and AM260W reaction centres, and the possible implications for the evolution of reaction centres as bioenergetic complexes are considered.
Asunto(s)
Evolución Molecular , Modelos Moleculares , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/efectos de la radiación , Rhodobacter sphaeroides/química , Rhodobacter sphaeroides/efectos de la radiación , Ubiquinona/química , Cristalografía por Rayos X , Luz , Mutación , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/efectos de la radiación , Rhodobacter sphaeroides/genética , Relación Estructura-ActividadRESUMEN
Photo-excitation of membrane-bound Rhodobacter sphaeroides reaction centres containing the mutation Ala M260 to Trp (AM260W) resulted in the accumulation of a radical pair state involving the photo-oxidised primary electron donor (P). This state had a lifetime of hundreds of milliseconds and its formation was inhibited by stigmatellin. The absence of the Q(A) ubiquinone in the AM260W reaction centre suggests that this long-lived radical pair state is P(+)Q(B)(-), although the exact reduction/protonation state of the Q(B) quinone remains to be confirmed. The blockage of active branch (A-branch) electron transfer by the AM260W mutation implies that this P(+)Q(B)(-) state is formed by electron transfer along the so-called inactive branch (B-branch) of reaction centre cofactors. We discuss how further mutations may affect the yield of the P(+)Q(B)(-) state, including a double alanine mutation (EL212A/DL213A) that probably has a direct effect on the efficiency of the low yield electron transfer step from the anion of the B-branch bacteriopheophytin (H(B)(-)) to the Q(B) ubiquinone.