RESUMEN
Plant photosynthetic (thylakoid) membranes are organized into complex networks that are differentiated into 2 distinct morphological and functional domains called grana and stroma lamellae. How the 2 domains join to form a continuous lamellar system has been the subject of numerous studies since the mid-1950s. Using different electron tomography techniques, we found that the grana and stroma lamellae are connected by an array of pitch-balanced right- and left-handed helical membrane surfaces of different radii and pitch. Consistent with theoretical predictions, this arrangement is shown to minimize the surface and bending energies of the membranes. Related configurations were proposed to be present in the rough endoplasmic reticulum and in dense nuclear matter phases theorized to exist in neutron star crusts, where the right- and left-handed helical elements differ only in their handedness. Pitch-balanced helical elements of alternating handedness may thus constitute a fundamental geometry for the efficient packing of connected layers or sheets.
Asunto(s)
Lactuca/ultraestructura , Tilacoides/ultraestructura , Tomografía con Microscopio Electrónico , Retículo Endoplásmico/ultraestructura , Lactuca/metabolismo , FotosíntesisRESUMEN
Members of the ß-karyopherin family mediate nuclear import of ribosomal proteins and export of ribosomal subunits, both required for ribosome biogenesis. We report that transcription of the ß-karyopherin genes importin 7 (IPO7) and exportin 1 (XPO1), and several additional nuclear import receptors, is regulated positively by c-Myc and negatively by p53. Partial IPO7 depletion triggers p53 activation and p53-dependent growth arrest. Activation of p53 by IPO7 knockdown has distinct features of ribosomal biogenesis stress, with increased binding of Mdm2 to ribosomal proteins L5 and L11 (RPL5 and RPL11). Furthermore, p53 activation is dependent on RPL5 and RPL11. Of note, IPO7 and XPO1 are frequently overexpressed in cancer. Altogether, we propose that c-Myc and p53 counter each other in the regulation of elements within the nuclear transport machinery, thereby exerting opposing effects on the rate of ribosome biogenesis. Perturbation of this balance may play a significant role in promoting cancer.
Asunto(s)
Carioferinas/fisiología , Proteínas Proto-Oncogénicas c-myc/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Ribosomas/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Transporte Activo de Núcleo Celular , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Carioferinas/genética , Carioferinas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Ribosómicas/metabolismo , Estrés Fisiológico , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína Exportina 1RESUMEN
In this paper we propose an energy dissipation mechanism that is completely reliant on changes in the aggregation state of the phycobilisome light-harvesting antenna components. All photosynthetic organisms regulate the efficiency of excitation energy transfer (EET) to fit light energy supply to biochemical demands. Not many do this to the extent required of desert crust cyanobacteria. Following predawn dew deposition, they harvest light energy with maximum efficiency until desiccating in the early morning hours. In the desiccated state, absorbed energy is completely quenched. Time and spectrally resolved fluorescence emission measurements of the desiccated desert crust Leptolyngbya ohadii strain identified (i) reduced EET between phycobilisome components, (ii) shorter fluorescence lifetimes, and (iii) red shift in the emission spectra, compared with the hydrated state. These changes coincide with a loss of the ordered phycobilisome structure, evident from small-angle neutron and X-ray scattering and cryo-transmission electron microscopy data. Based on these observations we propose a model where in the hydrated state the organized rod structure of the phycobilisome supports directional EET to reaction centers with minimal losses due to thermal dissipation. In the desiccated state this structure is lost, giving way to more random aggregates. The resulting EET path will exhibit increased coupling to the environment and enhanced quenching.
Asunto(s)
Cianobacterias/fisiología , Clima Desértico , Microbiología del Suelo , Complejos de Proteína Captadores de Luz , Fotosíntesis/fisiología , Ficobilisomas/fisiologíaRESUMEN
FtsZ proteins of the FtsZ1 and FtsZ2 families play important roles in the initiation and progression of plastid division in plants and green algae. Arabidopsis possesses a single FTSZ1 member and two FTSZ2 members, FTSZ2-1 and FTSZ2-2. The contribution of these to chloroplast division and partitioning has been mostly investigated in leaf mesophyll tissues. Here, we assessed the involvement of the three FtsZs in plastid division at earlier stages of chloroplast differentiation. To this end, we studied the effect of the absence of specific FtsZ proteins on plastids in the vegetative shoot apex, where the proplastid-to-chloroplast transition takes place. We found that the relative contribution of the two major leaf FtsZ isoforms, FtsZ1 and FtsZ2-1, to the division process varies with cell lineage and position within the shoot apex. While FtsZ2-1 dominates division in the L1 and L3 layers of the shoot apical meristem (SAM), in the L2 layer, FtsZ1 and FtsZ2-1 contribute equally toward the process. Depletion of the third isoform, FtsZ2-2, generally resulted in stronger effects in the shoot apex than those observed in mature leaves. The implications of these findings, along with additional observations made in this work, to our understanding of the mechanisms and regulation of plastid proliferation in the shoot apex are discussed.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Meristema/metabolismo , Hojas de la Planta/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cloroplastos/genética , Meristema/genética , Hojas de la Planta/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
Two LHC-like proteins, Photosystem II Subunit S (PSBS) and Light-Harvesting Complex Stress-Related (LHCSR), are essential for triggering excess energy dissipation in chloroplasts of vascular plants and green algae, respectively. The mechanism of quenching was studied in Physcomitrella patens, an early divergent streptophyta (including green algae and land plants) in which both proteins are active. PSBS was localized in grana together with photosystem II (PSII), but LHCSR was located mainly in stroma-exposed membranes together with photosystem I (PSI), and its distribution did not change upon high-light treatment. The quenched conformation can be preserved by rapidly freezing the high-light-treated tissues in liquid nitrogen. When using green fluorescent protein as an internal standard, 77K fluorescence emission spectra on isolated chloroplasts allowed for independent assessment of PSI and PSII fluorescence yield. Results showed that both photosystems underwent quenching upon high-light treatment in the wild type in contrast to mutants depleted of LHCSR, which lacked PSI quenching. Due to the contribution of LHCII, P. patens had a PSI antenna size twice as large with respect to higher plants. Thus, LHCII, which is highly abundant in stroma membranes, appears to be the target of quenching by LHCSR.
Asunto(s)
Bryopsida/metabolismo , Complejos de Proteína Captadores de Luz/metabolismo , Complejo de Proteína del Fotosistema I/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Proteínas de Plantas/metabolismo , Estrés Fisiológico , Bryopsida/efectos de los fármacos , Bryopsida/efectos de la radiación , Bryopsida/ultraestructura , Catálisis/efectos de los fármacos , Clorofila/metabolismo , Cloroplastos/efectos de los fármacos , Cloroplastos/metabolismo , Cloroplastos/ultraestructura , Digitonina/farmacología , Glucósidos/farmacología , Luz , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Microdominios de Membrana/efectos de la radiación , Procesos Fotoquímicos/efectos de los fármacos , Espectrometría de Fluorescencia , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/efectos de la radiación , Termodinámica , Tilacoides/metabolismo , Tilacoides/efectos de la radiación , Tilacoides/ultraestructuraRESUMEN
The cyanobacterium Synechocystis PCC 6803 possesses three Rieske isoforms: PetC1, PetC2 and PetC3. While PetC1 and PetC2 have been identified as alternative subunits of the cytochrome b6f complex (b6f), PetC3 was localized exclusively within the plasma membrane. The spatial separation of PetC3 from the photosynthetic and respiratory protein complexes raises doubt in its involvement in bioenergetic electron transfer. Here we report a detailed structural and functional characterization of the cyanobacterial PetC3 protein family indicating that PetC3 is not a component of the b6f and the photosynthetic electron transport as implied by gene annotation. Instead PetC3 has a distinct function in cell envelope homeostasis. Especially proteomic analysis shows that deletion of petC3 in Synechocystis PCC 6803 primarily affects cell envelope proteins including many nutrient transport systems. Therefore, the observed downregulation in the photosynthetic electron transport - mainly caused by photosystem 2 inactivation - might constitute a stress adaptation. Comprehensive in silico sequence analyses revealed that PetC3 proteins are periplasmic lipoproteins tethered to the plasma membrane with a subclass consisting of soluble periplasmic proteins, i.e. their N-terminal domain is inconsistent with their integration into the b6f. For the first time, the structure of PetC3 was determined by X-ray crystallography at an atomic resolution revealing significant high similarities to non-b6f Rieske subunits in contrast to PetC1. These results suggest that PetC3 affects processes in the periplasmic compartment that only indirectly influence photosynthetic electron transport. For this reason, we suggest to rename "Photosynthetic electron transport Chain 3" (PetC3) proteins as "periplasmic Rieske proteins" (Prp).
Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Fotosíntesis , Synechocystis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Transporte de Electrón , Proteínas del Complejo de Cadena de Transporte de Electrón/química , Proteínas del Complejo de Cadena de Transporte de Electrón/genética , Transferencia de Energía , Homeostasis , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Modelos Moleculares , Periplasma/metabolismo , Filogenia , Dominios y Motivos de Interacción de Proteínas , Proteómica , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Synechocystis/genética , Synechocystis/crecimiento & desarrolloRESUMEN
Over-reduction of the photosynthetic electron transport chain may severely damage the photosynthetic apparatus as well as other constituents of the chloroplast and the cell. Here, we exposed Arabidopsis leaves to saturating light either under normal atmospheric conditions or under CO2--and O2 -limiting conditions, which greatly increase excitation and electron pressures by draining terminal electron acceptors. The two treatments were found to have very different, often opposing, effects on the structure of the thylakoid membranes, including the width of the granal lumenal compartment. Modulation of the latter is proposed to be related to movements of ions across the thylakoid membrane, which alter the relative osmolarity of the lumen and stroma and affect the partitioning of the proton motive force into its electrical and osmotic components. The resulting changes in thylakoid organization and lumenal width should facilitate the repair of photodamaged photosystem II complexes in response to light stress under ambient conditions, but are expected to inhibit the repair cycle when the light stress occurs concurrently with CO2 and O2 depletion. Under the latter conditions, the changes in thylakoid structure are predicted to complement other processes that restrict the flow of electrons into the high-potential chain, thus moderating the production of deleterious reactive oxygen species at photosystem I.
Asunto(s)
Arabidopsis/fisiología , Dióxido de Carbono/metabolismo , Oxígeno/metabolismo , Tilacoides/fisiología , Arabidopsis/efectos de la radiación , Arabidopsis/ultraestructura , Transporte de Electrón , Luz , Estrés Oxidativo , Fotosíntesis/fisiología , Complejo de Proteína del Fotosistema I/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Hojas de la Planta/fisiología , Hojas de la Planta/efectos de la radiación , Hojas de la Planta/ultraestructura , Tilacoides/efectos de la radiación , Tilacoides/ultraestructuraRESUMEN
Biological desert sand crusts are the foundation of desert ecosystems, stabilizing the sands and allowing colonization by higher order organisms. The first colonizers of the desert sands are cyanobacteria. Facing the harsh conditions of the desert, these organisms must withstand frequent desiccation-hydration cycles, combined with high light intensities. Here, we characterize structural and functional modifications to the photosynthetic apparatus that enable a cyanobacterium, Leptolyngbya sp., to thrive under these conditions. Using multiple in vivo spectroscopic and imaging techniques, we identified two complementary mechanisms for dissipating absorbed energy in the desiccated state. The first mechanism involves the reorganization of the phycobilisome antenna system, increasing excitonic coupling between antenna components. This provides better energy dissipation in the antenna rather than directed exciton transfer to the reaction center. The second mechanism is driven by constriction of the thylakoid lumen which limits diffusion of plastocyanin to P700. The accumulation of P700(+) not only prevents light-induced charge separation but also efficiently quenches excitation energy. These protection mechanisms employ existing components of the photosynthetic apparatus, forming two distinct functional modes. Small changes in the structure of the thylakoid membranes are sufficient for quenching of all absorbed energy in the desiccated state, protecting the photosynthetic apparatus from photoinhibitory damage. These changes can be easily reversed upon rehydration, returning the system to its high photosynthetic quantum efficiency.
RESUMEN
During desiccation, homoiochlorophyllous resurrection plants retain most of their photosynthetic apparatus, allowing them to resume photosynthetic activity quickly upon water availability. These plants rely on various mechanisms to prevent the formation of reactive oxygen species and/or protect their tissues from the damage they inflict. In this work, we addressed the issue of how homoiochlorophyllous resurrection plants deal with the problem of excessive excitation/electron pressures during dehydration using Craterostigma pumilum as a model plant. To investigate the alterations in the supramolecular organization of photosynthetic protein complexes, we examined cryoimmobilized, freeze-fractured leaf tissues using (cryo)scanning electron microscopy. These examinations revealed rearrangements of photosystem II (PSII) complexes, including a lowered density during moderate dehydration, consistent with a lower level of PSII proteins, as shown by biochemical analyses. The latter also showed a considerable decrease in the level of cytochrome f early during dehydration, suggesting that initial regulation of the inhibition of electron transport is achieved via the cytochrome b6f complex. Upon further dehydration, PSII complexes are observed to arrange into rows and semicrystalline arrays, which correlates with the significant accumulation of sucrose and the appearance of inverted hexagonal lipid phases within the membranes. As opposed to PSII and cytochrome f, the light-harvesting antenna complexes of PSII remain stable throughout the course of dehydration. Altogether, these results, along with photosynthetic activity measurements, suggest that the protection of retained photosynthetic components is achieved, at least in part, via the structural rearrangements of PSII and (likely) light-harvesting antenna complexes into a photochemically quenched state.
Asunto(s)
Craterostigma/fisiología , Fotosíntesis/fisiología , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Craterostigma/genética , Craterostigma/efectos de la radiación , Complejo de Citocromo b6f/genética , Complejo de Citocromo b6f/metabolismo , Deshidratación , Desecación , Transporte de Electrón , Luz , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Hojas de la Planta/genética , Hojas de la Planta/fisiología , Hojas de la Planta/efectos de la radiación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Agua/fisiologíaRESUMEN
The chromatophores of Rhodobacter (Rb.) sphaeroides represent a minimal bio-energetic system, which efficiently converts light energy into usable chemical energy. Despite extensive studies, several issues pertaining to the morphology and molecular architecture of this elemental energy conversion system remain controversial or unknown. To tackle these issues, we combined electron microscope tomography, immuno-electron microscopy and atomic force microscopy. We found that the intracellular Rb. sphaeroides chromatophores form a continuous reticulum rather than existing as discrete vesicles. We also found that the cytochrome bc1 complex localizes to fragile chromatophore regions, which most likely constitute the tubular structures that interconnect the vesicles in the reticulum. In contrast, the peripheral light-harvesting complex 2 (LH2) is preferentially hexagonally packed within the convex vesicular regions of the membrane network. Based on these observations, we propose that the bc1 complexes are in the inter-vesicular regions and surrounded by reaction center (RC) core complexes, which in turn are bounded by arrays of peripheral antenna complexes. This arrangement affords rapid cycling of electrons between the core and bc1 complexes while maintaining efficient excitation energy transfer from LH2 domains to the RCs.
Asunto(s)
Cromatóforos/ultraestructura , Transferencia de Energía/genética , Fotosíntesis , Rhodobacter sphaeroides/metabolismo , Cromatóforos/química , Cromatóforos/metabolismo , Citoplasma/metabolismo , Luz , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/ultraestructura , Microscopía de Fuerza Atómica , Rhodobacter sphaeroides/crecimiento & desarrolloRESUMEN
Chloroplasts of higher plants develop from proplastids, which are undifferentiated plastids that lack photosynthetic (thylakoid) membranes. In flowering plants, the proplastid-chloroplast transition takes place at the shoot apex, which consists of the shoot apical meristem (SAM) and the flanking leaf primordia. It has been believed that the SAM contains only proplastids and that these become chloroplasts only in the primordial leaves. Here, we show that plastids of the SAM are neither homogeneous nor necessarily null. Rather, their developmental state varies with the specific region and/or layer of the SAM in which they are found. Plastids throughout the L1 and L3 layers of the SAM possess fairly developed thylakoid networks. However, many of these plastids eventually lose their thylakoids during leaf maturation. By contrast, plastids at the central, stem cell-harboring region of the L2 layer of the SAM lack thylakoid membranes; these appear only at the periphery, near the leaf primordia. Thus, plastids in the SAM undergo distinct differentiation processes that, depending on their lineage and position, lead to either development or loss of thylakoid membranes. These processes continue along the course of leaf maturation.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Brotes de la Planta/crecimiento & desarrollo , Plastidios/metabolismo , Tilacoides/metabolismo , Meristema/crecimiento & desarrollo , Microscopía , Plastidios/ultraestructuraRESUMEN
The machinery that conducts the light-driven reactions of oxygenic photosynthesis is hosted within specialized paired membranes called thylakoids. In higher plants, the thylakoids are segregated into two morphological and functional domains called grana and stroma lamellae. A large fraction of the luminal volume of the granal thylakoids is occupied by the oxygen-evolving complex of photosystem II. Electron microscopy data we obtained on dark- and light-adapted Arabidopsis thylakoids indicate that the granal thylakoid lumen significantly expands in the light. Models generated for the organization of the oxygen-evolving complex within the granal lumen predict that the light-induced expansion greatly alleviates restrictions imposed on protein diffusion in this compartment in the dark. Experiments monitoring the redox kinetics of the luminal electron carrier plastocyanin support this prediction. The impact of the increase in protein mobility within the granal luminal compartment in the light on photosynthetic electron transport rates and processes associated with the repair of photodamaged photosystem II complexes is discussed.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Cloroplastos/metabolismo , Tilacoides/metabolismo , Arabidopsis/ultraestructura , Citocromos f/metabolismo , Oscuridad , Difusión , Cinética , Modelos Biológicos , Oxidación-Reducción , Tilacoides/ultraestructuraRESUMEN
The balance between linear electron transport (LET) and cyclic electron transport (CET) plays an essential role in plant adaptation and protection against photo-induced damage. This balance is largely maintained by phosphorylation-driven alterations in the PSII-LHCII assembly and thylakoid membrane stacking. During the dark-to-light transition, plants shift this balance from CET, which prevails to prevent overreduction of the electron transport chain and consequent photo-induced damage, towards LET, which enables efficient CO2 assimilation and biomass production. Using freeze-fracture cryo-scanning electron microscopy and transmission electron microscopy of Arabidopsis leaves, we reveal unique membrane regions possessing characteristics of both stacked and unstacked regions of the thylakoid network that form during this transition. A notable consequence of the morphological attributes of these regions, which we refer to as 'stacked thylakoid doublets', is an overall increase in the proximity and connectivity of the two photosystems (PSI and PSII) that drive LET. This, in turn, reduces diffusion distances and barriers for the mobile carriers that transfer electrons between the two PSs, thereby maximizing LET and optimizing the plant's ability to utilize light energy. The mechanics described here for the shift between CET and LET during the dark-to-light transition are probably also used during chromatic adaptation mediated by state transitions.
Asunto(s)
Arabidopsis , Tilacoides , Tilacoides/metabolismo , Transporte de Electrón , Complejo de Proteína del Fotosistema I/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Electrones , Complejos de Proteína Captadores de Luz/metabolismo , Arabidopsis/metabolismo , Luz , FotosíntesisRESUMEN
Ricin, a highly potent plant-derived toxin, is considered a potential bioterrorism weapon due to its pronounced toxicity, high availability, and ease of preparation. Acute damage following pulmonary ricinosis is characterized by local cytokine storm, massive neutrophil infiltration, and edema formation, resulting in respiratory insufficiency and death. A designated equine polyclonal antibody-based (antitoxin) treatment was developed in our laboratory and proved efficacious in alleviating lung injury and increasing survival rates. Although short-term pathogenesis was thoroughly characterized in antitoxin-treated mice, the long-term damage in surviving mice was never determined. In this study, long-term consequences of ricin intoxication were evaluated 30 days post-exposure in mice that survived antitoxin treatment. Significant pulmonary sequelae were demonstrated in surviving antitoxin-treated mice, as reflected by prominent histopathological changes, moderate fibrosis, increased lung hyperpermeability, and decreased lung compliance. The presented data highlight, for the first time to our knowledge, the possibility of long-term damage development in mice that survived lethal-dose pulmonary exposure to ricin due to antitoxin treatment.
Asunto(s)
Antitoxinas , Lesión Pulmonar , Insuficiencia Respiratoria , Ricina , Animales , Caballos , Ratones , Antitoxinas/uso terapéutico , Ricina/toxicidad , Pulmón/patología , Lesión Pulmonar/tratamiento farmacológicoRESUMEN
The process of oxygenic photosynthesis enabled and still sustains aerobic life on Earth. The most elaborate form of the apparatus that carries out the primary steps of this vital process is the one present in higher plants. Here, we review the overall composition and supramolecular organization of this apparatus, as well as the complex architecture of the lamellar system within which it is harbored. Along the way, we refer to the genetic, biochemical, spectroscopic and, in particular, microscopic studies that have been employed to elucidate the structure and working of this remarkable molecular energy conversion device. As an example of the highly dynamic nature of the apparatus, we discuss the molecular and structural events that enable it to maintain high photosynthetic yields under fluctuating light conditions. We conclude the review with a summary of the hypotheses made over the years about the driving forces that underlie the partition of the lamellar system of higher plants and certain green algae into appressed and non-appressed membrane domains and the segregation of the photosynthetic protein complexes within these domains.
Asunto(s)
Embryophyta/fisiología , Fotosíntesis , Proteínas del Complejo del Centro de Reacción Fotosintética/fisiología , Tilacoides/fisiología , Luz , FosforilaciónRESUMEN
Exposure of cyanobacterial or red algal cells to high light has been proposed to lead to excitonic decoupling of the phycobilisome antennae (PBSs) from the reaction centers. Here we show that excitonic decoupling of PBSs of Synechocystis sp. PCC 6803 is induced by strong light at wavelengths that excite either phycobilin or chlorophyll pigments. We further show that decoupling is generally followed by disassembly of the antenna complexes and/or their detachment from the thylakoid membrane. Based on a previously proposed mechanism, we suggest that local heat transients generated in the PBSs by non-radiative energy dissipation lead to alterations in thermo-labile elements, likely in certain rod and core linker polypeptides. These alterations disrupt the transfer of excitation energy within and from the PBSs and destabilize the antenna complexes and/or promote their dissociation from the reaction centers and from the thylakoid membranes. Possible implications of the aforementioned alterations to adaptation of cyanobacteria to light and other environmental stresses are discussed.
Asunto(s)
Cianobacterias , Luz , Ficobilisomas/química , Ficobilisomas/fisiología , Ficobilisomas/efectos de la radiación , Estrés Fisiológico/fisiología , Cianobacterias/metabolismo , Cianobacterias/ultraestructura , Transporte de Electrón/efectos de la radiación , Recuperación de Fluorescencia tras Fotoblanqueo , Microscopía Confocal , Modelos Biológicos , Multimerización de Proteína/efectos de la radiación , Estructura Cuaternaria de Proteína , Espectrometría de Fluorescencia , Estrés Fisiológico/efectos de la radiación , Synechocystis/metabolismo , Synechocystis/fisiología , Synechocystis/ultraestructura , TemperaturaRESUMEN
Mutations in a protein active site can lead to dramatic and useful changes in protein activity. The active site, however, is sensitive to mutations due to a high density of molecular interactions, substantially reducing the likelihood of obtaining functional multipoint mutants. We introduce an atomistic and machine-learning-based approach, called high-throughput Functional Libraries (htFuncLib), that designs a sequence space in which mutations form low-energy combinations that mitigate the risk of incompatible interactions. We apply htFuncLib to the GFP chromophore-binding pocket, and, using fluorescence readout, recover >16,000 unique designs encoding as many as eight active-site mutations. Many designs exhibit substantial and useful diversity in functional thermostability (up to 96 °C), fluorescence lifetime, and quantum yield. By eliminating incompatible active-site mutations, htFuncLib generates a large diversity of functional sequences. We envision that htFuncLib will be used in one-shot optimization of activity in enzymes, binders, and other proteins.
Asunto(s)
Proteínas , Dominio Catalítico , Biblioteca de Genes , Proteínas/genética , Mutación , Fluorescencia , Proteínas Fluorescentes Verdes/metabolismoRESUMEN
Polypyrimidine tract binding protein 1 (PTBP1) is thought to be expressed only at embryonic stages in central neurons. Its down-regulation triggers neuronal differentiation in precursor and non-neuronal cells, an approach recently tested for generation of neurons de novo for amelioration of neurodegenerative disorders. Moreover, PTBP1 is replaced by its paralog PTBP2 in mature central neurons. Unexpectedly, we found that both proteins are coexpressed in adult sensory and motor neurons, with PTBP2 restricted mainly to the nucleus, while PTBP1 also shows axonal localization. Levels of axonal PTBP1 increased markedly after peripheral nerve injury, and it associates in axons with mRNAs involved in injury responses and nerve regeneration, including importin ß1 (KPNB1) and RHOA. Perturbation of PTBP1 affects local translation in axons, nociceptor neuron regeneration and both thermal and mechanical sensation. Thus, PTBP1 has functional roles in adult axons. Hence, caution is required before considering targeting of PTBP1 for therapeutic purposes.
Asunto(s)
Axones , Regeneración Nerviosa , Neuronas , Traumatismos de los Nervios Periféricos , Adulto , Humanos , Axones/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Interneuronas/metabolismo , Regeneración Nerviosa/genética , Neuronas/metabolismo , Traumatismos de los Nervios Periféricos/genética , Traumatismos de los Nervios Periféricos/metabolismoRESUMEN
The COVID-19 pandemic caused by the SARS-CoV-2 infection induced lung inflammation characterized by cytokine storm and fulminant immune response of both resident and migrated immune cells, accelerating alveolar damage. In this work we identified members of the matrix metalloprotease (MMPs) family associated with lung extra-cellular matrix (ECM) destruction using K18-hACE2-transgenic mice (K18-hACE2) infected intranasally with SARS-CoV-2. Five days post infection, the lungs exhibited overall alveolar damage of epithelial cells and massive leukocytes infiltration. A substantial pulmonary increase in MMP8, MMP9, and MMP14 in the lungs post SARS-CoV-2 infection was associated with degradation of ECM components including collagen, laminin, and proteoglycans. The process of tissue damage and ECM degradation during SARS-CoV-2 lung infection is suggested to be associated with activity of members of the MMPs family, which in turn may be used as a therapeutic intervention.
Asunto(s)
COVID-19 , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2 , Animales , Modelos Animales de Enfermedad , Humanos , Pulmón/patología , Melfalán , Ratones , Ratones Transgénicos , Pandemias , Peptidil-Dipeptidasa A/metabolismo , gammaglobulinasRESUMEN
Tumors initiate by mutations in cancer cells, and progress through interactions of the cancer cells with non-malignant cells of the tumor microenvironment. Major players in the tumor microenvironment are cancer-associated fibroblasts (CAFs), which support tumor malignancy, and comprise up to 90% of the tumor mass in pancreatic cancer. CAFs are transcriptionally rewired by cancer cells. Whether this rewiring is differentially affected by different mutations in cancer cells is largely unknown. Here we address this question by dissecting the stromal landscape of BRCA-mutated and BRCA Wild-type pancreatic ductal adenocarcinoma. We comprehensively analyze pancreatic cancer samples from 42 patients, revealing different CAF subtype compositions in germline BRCA-mutated vs. BRCA Wild-type tumors. In particular, we detect an increase in a subset of immune-regulatory clusterin-positive CAFs in BRCA-mutated tumors. Using cancer organoids and mouse models we show that this process is mediated through activation of heat-shock factor 1, the transcriptional regulator of clusterin. Our findings unravel a dimension of stromal heterogeneity influenced by germline mutations in cancer cells, with direct implications for clinical research.