Search Results (24)

Fruit shape is an important quality and yield trait of pear, and the fruit shape of ‘Laiyang Cili’ presents a spindle shape which seriously affects its commercial value. Calyx excision treatment could change the fruit shape, while the underlying genes and their regulatory mechanism remain poorly understood. In this study, we constructed RNA-seq libraries of pear treated with calyx excision to explore underlying regulatory mechanisms. At the early stage of the calyx excision treatment, the numbers of differentially expressed genes (DEGs) between each comparison group were relatively high and gradually decreased along with fruit development. The expression pattern of the DEGs ranked in the top 30 of the six groups had obvious divergence, and DEGs were mainly distributed in the “after calyx excision treatment (0 days)” (AC0d) and AC2d groups. The DEGs were mainly enriched in plant hormone signal transduction and plant defense response. We identified 17 candidate genes related to fruit shape and tested their expression patterns along with fruit development. Among them, nine candidate genes expression trends were consistent with fragments per kilobase of exon model per million mapped fragment (FPKM) values, including MYB62, outer envelope pore protein 62 (OEP62), auxin response factor 3 (ARF3), auxin-responsive protein 50 (SAUR50), protein phosphatase 2C 51 (PP2C 51), major allergen Pyr c 1 (PYRC1), aquaporin TIP1-3 (TIP1-3), transcription factor TGA4 (TGA4) and auxin-responsive protein 17 (IAA17). And then, weighted gene co-expression network analysis (WGCNA) analysis revealed that the OVATE family protein (OFP) and SUN domain-containing protein (SUN) were divided into the MEblue model, which had a positive correlation with calyx excision treatment, and the expression trend of LOC103960706 (OFP8) appeared cohesive with FPKM values. Pbr014104.1 and Pbr016952.1, which were the ortholog genes of LOC103960706, were further identified from the pear genome, and were found to be highly expressed in pear fruit through RT-PCR analysis. Taken together, the key stage determining the development of fruit shape was in the early stage after calyx excision treatment, and fruit shape regulation and development were co-regulated by multiple genes. Full article

Gram-negative bacteria release outer membrane vesicles (OMVs) that deliver various molecules, including virulence factors, to interact with their host. Recent studies have suggested that OMVs may also serve as carriers for RNAs, particularly small regulatory noncoding RNAs (sRNAs). For these RNAs to function effectively, they typically require a protein cofactor, Hfq, known as an RNA chaperone. In previous work, using molecular imaging, Circular Dichroism CD, and InfraRed FTIR spectroscopies, we demonstrated that Hfq interacts with the bacterial inner membrane and forms pores, suggesting a possible role in translocating RNA from the cytoplasm to periplasm and then to OMVs. In this study, we expand on our previous findings and provide evidence that RNA molecules bind to the Escherichia coli inner membrane in an Hfq-dependent manner. Moreover, we show that the lipid nature, in particular the presence of a cardiolipin-rich domain, is crucial for this interaction. These results reveal a new aspect of RNA translocation through the inner membrane, for further packaging in OMVs, and underscore the importance of Hfq in this mechanism. Full article

The outer membrane of Gram-negative bacteria contains a variety of pore-forming structures collectively referred to as porins. Some of these are voltage dependent, but weakly so, closing at high voltages. Triplin, a novel bacterial pore-former, is a three-pore structure, highly voltage dependent, with a complex gating process. The three pores close sequentially: pore 1 at positive potentials, 2 at negative and 3 at positive. A positive domain containing 14 positive charges (the voltage sensor) translocates through the membrane during the closing process, and the translocation is proposed to take place by the domain entering the pore and thus blocking it, resulting in the closed conformation. This mechanism of pore closure is supported by kinetic measurements that show that in the closing process the voltage sensor travels through most of the transmembrane voltage before reaching the energy barrier. Voltage-dependent blockage of the pores by polyarginine, but not by a 500-fold higher concentrations of polylysine, is consistent with the model of pore closure, with the sensor consisting mainly of arginine residues, and with the presence, in each pore, of a complementary surface that serves as a binding site for the sensor. Full article

Nuclear protein prothymosin α (ProTα) is a unique member of damage-associated molecular patterns (DAMPs)/alarmins. ProTα prevents neuronal necrosis by causing a cell death mode switch in serum-starving or ischemic/reperfusion models in vitro and in vivo. Underlying receptor mechanisms include Toll-like receptor 4 (TLR4) and G_i-coupled receptor. Recent studies have revealed that the mode of the fatal stress-induced extracellular release of nuclear ProTα from cortical neurons in primary cultures, astrocytes and C6 glioma cells has two steps: ATP loss-induced nuclear release and the Ca²⁺-mediated formation of a multiple protein complex and its extracellular release. Under the serum-starving condition, ProTα is diffused from the nucleus throughout the cell due to the ATP loss-induced impairment of importin α–mediated nuclear transport. Subsequent mechanisms are all Ca²⁺-dependent. They include the formation of a protein complex with ProTα, S100A13, p40 Syt-1 and Annexin A2 (ANXA2); the fusion of the protein complex to the plasma membrane via p40 Syt-1–Stx-1 interaction; and TMEM16F scramblase-mediated ANXA2 flop-out. Subsequently, the protein complex is extracellularly released, leaving ANXA2 on the outer cell surface. The ANXA2 is then flipped in by a force of ATP8A2 activity, and the non-vesicular release of protein complex is repeated. Thus, the ANXA2 flop-out could play key roles in a new type of non-vesicular and non-classical release for DAMPs/alarmins, which is distinct from the modes conducted via gasdermin D or mixed-lineage kinase domain-like pseudokinase pores. Full article

Apoptosis and necroptosis overlap in their initial signaling but diverge to produce non-inflammatory and pro-inflammatory outcomes, respectively. High glucose pushes signaling in favor of necroptosis producing a hyperglycemic shift from apoptosis to necroptosis. This shift depends on receptor-interacting protein 1 (RIP1) and mitochondrial reactive oxygen species (ROS). Here, we show that RIP1, mixed lineage kinase domain-like (MLKL) protein, Bcl-2 agonist/killer (Bak), Bcl-2 associated x (Bax) protein, and dynamin-related protein 1 (Drp1) traffic to the mitochondria in high glucose. RIP1 and MLKL appear in the mitochondria in their activated, phosphorylated states while Drp1 appears in its activated, dephosphorylated state in high glucose. Mitochondrial trafficking is prevented in rip1 KO cells and upon treatment with N-acetylcysteine. Induction of ROS replicated the mitochondrial trafficking seen in high glucose. MLKL forms high MW oligomers in the outer and inner mitochondrial membranes while Bak and Bax form high MW oligomers in the outer mitochondrial membrane in high glucose, suggesting pore formation. MLKL, Bax, and Drp1 promoted cytochrome c release from the mitochondria as well as a decrease in mitochondrial membrane potential in high glucose. These results indicate that mitochondrial trafficking of RIP1, MLKL, Bak, Bax, and Drp1 are key events in the hyperglycemic shift from apoptosis to necroptosis. This is also the first report to show oligomerization of MLKL in the inner and outer mitochondrial membranes and dependence of mitochondrial permeability on MLKL. Full article

VDAC (Voltage-Dependent Anion-selective Channel) is the primary metabolite pore in the mitochondrial outer membrane (OM). Atomic structures of VDAC, consistent with its physiological “open” state, are β-barrels formed by 19 transmembrane (TM) β-strands and an N-terminal segment (NTERM) that folds inside the pore lumen. However, structures are lacking for VDAC’s partially “closed” states. To provide clues about possible VDAC conformers, we used the RoseTTAFold neural network to predict structures for human and fungal VDAC sequences modified to mimic removal from the pore wall or lumen of “cryptic” domains, i.e., segments buried in atomic models yet accessible to antibodies in OM-bound VDAC. Predicted in vacuo structures for full-length VDAC sequences are 19-strand β-barrels similar to atomic models, but with weaker H-bonding between TM strands and reduced interactions between NTERM and the pore wall. Excision of combinations of “cryptic” subregions yields β-barrels with smaller diameters, wide gaps between N- and C-terminal β-strands, and in some cases disruption of the β-sheet (associated with strained backbone H-bond registration). Tandem repeats of modified VDAC sequences also were explored, as was domain swapping in monomer constructs. Implications of the results for possible alternative conformational states of VDAC are discussed. Full article

A sodium channel blocker mexiletine (MEX) is used to treat chronic pain, myotonia and some arrhythmias. Mutations in the pore domain (PD) of voltage-gated sodium channels differently affect tonic block (TB) and use-dependent block (UDB) by MEX. Previous studies identified several MEX-sensing residues in the hNav1.5 channel and demonstrated that the channel block by MEX increases with activation of the voltage-sensing domain III (VSD_III), whereas MEX stabilizes the activated state of VSD_III. Structural rationales for these observations are unclear. Here, Monte Carlo (MC) energy minimizations were used to dock MEX and its more potent analog, Thio-Me2, into the hNav1.5 cryo-EM structure with activated VSDs and presumably inactivated PD. Computations yielded two ensembles of ligand binding poses in close contacts with known MEX-sensing residues in helices S6_III, S6_IV and P1_IV. In both ensembles, the ligand NH₃ group approached the cation-attractive site between backbone carbonyls at the outer-pore bottom, while the aromatic ring protruded ether into the inner pore (putative UDB pose) or into the III/IV fenestration (putative TB pose). In silico deactivation of VSD_III shifted helices S4–S5_III, S5_III, S6_III and S6_IV and tightened the TB site. In a model with activated VSD_III and three resting VSDs, MC-minimized energy profile of MEX pulled from the TB site towards lipids shows a deep local minimum due to interactions with 11 residues in S5_III, P1_III, S6_III and S6_IV. The minimum may correspond to an interim binding site for MEX in the hydrophobic path to the TB site along the lipid-exposed sides of repeats III and IV where 15 polar and aromatic residues would attract cationic blockers. The study explains numerous experimental data and suggests the mechanism of allosteric modification of the MEX binding site by VSD_III. Full article

Macrophage-expressed gene 1 proteins (Mpeg1/Perforin-2 (PRF2)) are a family of pore-forming proteins (PFPs) which can form pores and destroy the cell membrane of invading pathogens. However, little information is available regarding the function of Mpeg1 in the giant triton snail Charonia tritonis. In this study, a homolog of Mpeg1 (Ct-Mpeg1) was identified in C. tritonis. The predicted protein of Ct-Mpeg1 contains several structural features known in Mpegs, including a membrane attack complex/perforin (MACPF) domain and single transmembrane region. The Ct-Mpeg1 gene was constitutively expressed in almost all tissues examined except in the proboscis, with the highest expression level observed in the mantle. As a typical pore-forming protein, Ct-Mpeg1 has antibacterial activities against Vibrio (including Vibrio alginolyticus and Vibrio parahaemolyticus). In addition, rCt-Mpeg1 challenge to V. alginolyticus represses the expression of most outer membrane protein synthesis-related genes and genes involved in the TCA cycle pathway, which will lead to reduced outer membrane protein synthesis and less energy capacity. This is the first report to characterize the macrophage-expressed gene 1 protein in C. tritonis, and these results suggest that macrophage-expressed gene 1 protein Ct-Mpeg1 is an important immune molecule of C. tritonis that is involved in the bacterial infection resistance of Vibrio, and this study may provide crucial basic data for the understanding of the innate immunity system of C. tritonis. Full article

The pertussis agent Bordetella pertussis produces a number of virulence factors, of which the filamentous hemagglutinin (FhaB) plays a role in B. pertussis adhesion to epithelial and phagocytic cells. Moreover, FhaB was recently found to play a crucial role in nasal cavity infection and B. pertussis transmission to new hosts. The 367 kDa FhaB protein translocates through an FhaC pore to the outer bacterial surface and is eventually processed to a ~220 kDa N-terminal FHA fragment by the SphB1 protease. A fraction of the mature FHA then remains associated with bacterial cell surface, while most of FHA is shed into the bacterial environment. Previously reported indirect evidence suggested that FHA, or its precursor FhaB, may bind the β₂ integrin CD11b/CD18 of human macrophages. Therefore, we assessed FHA binding to various cells producing or lacking the integrin and show that purified mature FHA does not bind CD11b/CD18. Further results then revealed that the adhesion of B. pertussis to cells does not involve an interaction between the bacterial surface-associated FhaB and/or mature FHA and the β₂ integrin CD11b/CD18. In contrast, FHA binding was strongly inhibited at micromolar concentrations of heparin, corroborating that the cell binding of FHA is ruled by the interaction of its heparin-binding domain with sulfated glycosaminoglycans on the cell surface. Full article

For a long time, particular attention was paid to glauconitization in the surficial sediments lying on the outer continental shelves of present oceans. Subsequently, the processes observed and analyzed may have served as models for studies of glauconite in Cenozoic or even Mesozoic shelf deposits. Access to the sedimentary domains of deep oceans, particularly those of contouritic accumulation fields, has made it possible to discover unexpected processes of glauconitization. Thus, the long-term prevalence of control using fairly high-temperature water has become obsolete, and the prerequisite influence of continental flows has come to be considered on a new scale. Frequently, sediments from contouritic accumulation provide a condensed and undisturbed sedimentary record without periods of sediment erosion. Glauconitic grains could possibly integrate the signatures of bottom-water masses over prolonged periods of time, which, while preventing their use in high-resolution studies, would provide an effective means of yielding reliable average estimates on past εNd signatures of bottom-water masses. In this regard, glauconitic grains are probably better-suited to paleoceanographic reconstructions than foraminifera and leached Fe-oxyhydroxide fractions, which appear to be influenced by sediment redistribution and the presence of terrestrial continental Fe-oxides, respectively. Direct methodological access to the compositions of the semi-confined microenvironments of neoformation has largely renewed the information, chemical or crystallographic, that was previously, and for a long time, restricted to macromeasurements. The various granular supports (mudclasts, fecal pellets, and foraminifera infillings) include inherited 1:1 clays (or Te-Oc; i.e., clay minerals consisting of one tetrahedral sheet and one octahedral sheet, such as kaolinite) that are gradually replaced by 2:1 clays (Te-Oc-Te) dominated first by smectite, and then by glauconite. In small pores, the water’s activity is diminished; as a consequence, the precipitation of a great number of mineral species is thereby made easier, and their stability domains are changed. A specific methodological approach allows the study of the mineralogy and chemistry of the fine-scale mineral phases and to avoid the global aspect of the analytical methods previously used in the initial studies. Wide-field micrographs taken at a mean direct magnification of 100.000 show the intimate and characteristic organization of the main phases that occur in a single grain. One or several “fine” (about 10 nanometers in scale) microchemical analyses can be recorded, and directly coupled with each interesting and well-identified structure image observed in HRTEM. Full article

Bacteriophage M13 assembles its progeny particles in the inner membrane of the host. The major component of the assembly machine is G1p and together with G11p it generates an oligomeric structure with a pore-like inner cavity and an ATP hydrolysing domain. This allows the formation of the phage filament, which assembles multiple copies of the membrane-inserted major coat protein G8p around the extruding single-stranded circular DNA. The phage filament then passes through the G4p secretin that is localized in the outer membrane. Presumably, the inner membrane G1p/G11p and the outer G4p form a common complex. To unravel the structural details of the M13 assembly machine, we purified G1p from infected E. coli cells. The protein was overproduced together with G11p and solubilized from the membrane as a multimeric complex with a size of about 320 kDa. The complex revealed a pore-like structure with an outer diameter of about 12 nm, matching the dimensions of the outer membrane G4p secretin. The function of the M13 assembly machine for phage generation and secretion is discussed. Full article

The Bacteroidetes type IX secretion system (T9SS) consists of at least 20 components that translocate proteins with type A or type B C-terminal domain (CTD) signals across the outer membrane (OM). While type A CTD proteins are anchored to the cell surface via covalent linkage to the anionic lipopolysaccharide, it is still unclear how type B CTD proteins are anchored to the cell surface. Moreover, very little is known about the PorE and PorP components of the T9SS. In this study, for the first time, we identified a complex comprising the OM $\mathsf{\beta }$-barrel protein PorP, the OM-associated periplasmic protein PorE and the type B CTD protein PG1035. Cross-linking studies supported direct interactions between PorE-PorP and PorP-PG1035. Furthermore, we show that the formation of the PorE-PorP-PG1035 complex was independent of PorU and PorV. Additionally, the Flavobacterium johnsoniae PorP-like protein, SprF, was found bound to the major gliding motility adhesin, SprB, which is also a type B CTD protein. Together, these results suggest that type B-CTD proteins may anchor to the cell surface by binding to their respective PorP-like proteins. Full article

The optical filter based on the micro–nano structure on the material surface is an important optical device, which is widely used in many fields. The filter is fabricated on the substrate with different shapes and sizes of micro–nano array structure, and the wavelength selectivity is realized by adjusting the processing parameters. In this paper, the finite-difference time-domain (FDTD) method is used to simulate the spectral properties of periodic array structures on the Au surface, and the spectral response characteristics of different surface structural parameters to the incident light are obtained. The simulation results show that the periodic pore array has a directional modulation function on the reflectivity and transmittance of the material surface. In the same circular aperture array structure, the wavelength selection ability is proportional to the interval distance of the array period, but the transmission peak linewidth decreases with the increase of the interval distance. The structural spectrum of the cylindrical array is closely related to the structural period. The period of the array structure increases in proportion, the center wavelengths of the reflection and transmission peak of the spectrum are red-shifted. When the height of the array structure increases proportionally, the positions of the center wavelengths of the reflection and transmission peak remain almost unchanged. When the period of the array structure increases, the center wavelength of the reflection and transmission peaks appear red-shifted, and the line width is also narrowed. For the periodic ring array structure, as the inner diameter increases, the reflection peak is significantly red-shifted, and the smaller the ring width, the faster the red-shift of the reflection peak with the wavelength. By controlling the ratio of inner diameter-to-outer diameter, the spectral characteristics of the structured surface can be effectively controlled. These simulation results provide a basis for the preparation of optical filters in the future and a new idea for the study of micro–nano characteristic structures on the surface of materials. Full article

The voltage-dependent anion channel (VDAC) is a β-barrel membrane protein located in the outer mitochondrial membrane (OMM). VDAC has two conductance states: an open anion selective state, and a closed and slightly cation-selective state. VDAC conductance states play major roles in regulating permeability of ATP/ADP, regulation of calcium homeostasis, calcium flux within ER-mitochondria contact sites, and apoptotic signaling events. Three reported structures of VDAC provide information on the VDAC open state via X-ray crystallography and nuclear magnetic resonance (NMR). Together, these structures provide insight on how VDAC aids metabolite transport. The interaction partners of VDAC, together with the permeability of the pore, affect the molecular pathology of diseases including Parkinson’s disease (PD), Friedreich’s ataxia (FA), lupus, and cancer. To fully address the molecular role of VDAC in disease pathology, major questions must be answered on the structural conformers of VDAC. For example, further information is needed on the structure of the closed state, how binding partners or membrane potential could lead to the open/closed states, the function and mobility of the N-terminal α-helical domain of VDAC, and the physiological role of VDAC oligomers. This review covers our current understanding of the various states of VDAC, VDAC interaction partners, and the roles they play in mitochondrial regulation pertaining to human diseases. Full article

The voltage-dependent anion channel (VDAC) is the primary regulating pathway of water-soluble metabolites and ions across the mitochondrial outer membrane. When reconstituted into lipid membranes, VDAC responds to sufficiently large transmembrane potentials by transitioning to gated states in which ATP/ADP flux is reduced and calcium flux is increased. Two otherwise unrelated cytosolic proteins, tubulin, and α-synuclein (αSyn), dock with VDAC by a novel mechanism in which the transmembrane potential draws their disordered, polyanionic C-terminal domains into and through the VDAC channel, thus physically blocking the pore. For both tubulin and αSyn, the blocked state is observed at much lower transmembrane potentials than VDAC gated states, such that in the presence of these cytosolic docking proteins, VDAC’s sensitivity to transmembrane potential is dramatically increased. Remarkably, the features of the VDAC gated states relevant for bioenergetics—reduced metabolite flux and increased calcium flux—are preserved in the blocked state induced by either docking protein. The ability of tubulin and αSyn to modulate mitochondrial potential and ATP production in vivo is now supported by many studies. The common physical origin of the interactions of both tubulin and αSyn with VDAC leads to a general model of a VDAC inhibitor, facilitates predictions of the effect of post-translational modifications of known inhibitors, and points the way toward the development of novel therapeutics targeting VDAC. Full article