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排序方式: 共有937条查询结果,搜索用时 15 毫秒
81.
Zambelli B Stola M Musiani F De Vriendt K Samyn B Devreese B Van Beeumen J Turano P Dikiy A Bryant DA Ciurli S 《The Journal of biological chemistry》2005,280(6):4684-4695
Bacillus pasteurii UreG, a chaperone involved in the urease active site assembly, was overexpressed in Escherichia coli BL21(DE3) and purified to homogeneity. The identity of the recombinant protein was confirmed by SDS-PAGE, protein sequencing, and mass spectrometry. A combination of size exclusion chromatography and multiangle and dynamic laser light scattering established that BpUreG is present in solution as a dimer. Analysis of circular dichroism spectra indicated that the protein contains large portions of helices (15%) and strands (29%), whereas NMR spectroscopy indicated the presence of conformational fluxionality of the protein backbone in solution. BpUreG catalyzes the hydrolysis of GTP with a kcat=0.04 min(-1), confirming a role for this class of proteins in coupling energy requirements and nickel incorporation into the urease active site. BpUreG binds two Zn2+ ions per dimer, with a KD=42 +/- 3 microm, and has a 10-fold lower affinity for Ni2+. A structural model for BpUreG was calculated by using threading algorithms. The protein, in the fully folded state, features the typical structural architecture of GTPases, with an open beta-barrel surrounded by alpha-helices and a P-loop at the N terminus. The protein dynamic behavior observed in solution is critically discussed relative to the structural model, using algorithms for disorder predictions. The results suggest that UreG proteins belong to the class of intrinsically unstructured proteins that need the interaction with cofactors or other protein partners to perform their function. It is also proposed that metal ions such as Zn2+ could have important structural roles in the urease activation process. 相似文献
82.
Riedel CG Mazza M Maier P Körner R Knop M 《The Journal of biological chemistry》2005,280(45):37846-37852
During sporulation and meiosis of budding yeast a developmental program determines the formation of the new plasma membranes of the spores. This process of prospore membrane (PSM) formation leads to the formation of meiotic daughter cells, the spores, within the lumen of the mother cell. It is initiated at the spindle pole bodies during meiosis II. Spore formation, but not meiotic cell cycle progression, requires the function of phospholipase D (PLD/Spo14). Here we show that PLD/Spo14 forms a complex with Sma1, a meiotically expressed protein essential for spore formation. Detailed analysis revealed that both proteins are required for early steps of prospore membrane assembly but with distinct defects in the respective mutants. In the Deltaspo14 mutant the initiation of PSM formation is blocked and aggregated vesicles of homogenous size are detected at the spindle pole bodies. In contrast, initiation of PSM formation does occur in the Deltasma1 mutant, but the enlargement of the membrane is impaired. During PSM growth both Spo14 and Sma1 localize to the membrane, and localization of Spo14 is independent of Sma1. Biochemical analysis revealed that Sma1 is not necessary for PLD activity per se and that PLD present in a complex with Sma1 is highly active. Together, our results suggest that yeast PLD is involved in two distinct but essential steps during the regulated vesicle fusion necessary for the assembly of the membranous encapsulations of the spores. 相似文献
83.
Extended molecular dynamics simulation of the carbon monoxide migration in sperm whale myoglobin
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Bossa C Anselmi M Roccatano D Amadei A Vallone B Brunori M Di Nola A 《Biophysical journal》2004,86(6):3855-3862
We report the results of an extended molecular dynamics simulation on the migration of photodissociated carbon monoxide in wild-type sperm whale myoglobin. Our results allow following one possible ligand migration dynamics from the distal pocket to the Xe1 cavity via a path involving the other xenon binding cavities and momentarily two additional packing defects along the pathway. Comparison with recent time resolved structural data obtained by Laue crystallography with subnanosecond to millisecond resolution shows a more than satisfactory agreement. In fact, according to time resolved crystallography, CO, after photolysis, can occupy the Xe1 and Xe4 cavities. However, no information on the trajectory of the ligand from the distal pocket to the Xe1 is available. Our results clearly show one possible path within the protein. In addition, although our data refer to a single trajectory, the local dynamics of the ligand in each cavity is sufficiently equilibrated to obtain local structural and thermodynamic information not accessible to crystallography. In particular, we show that the CO motion and the protein fluctuations are strictly correlated: free energy calculations of the migration between adjacent cavities show that the migration is not a simple diffusion but is kinetically or thermodynamically driven by the collective motions of the protein; conversely, the protein fluctuations are influenced by the ligand in such a way that the opening/closure of the passage between adjacent cavities is strictly correlated to the presence of CO in its proximity. The compatibility between time resolved crystallographic experiments and molecular dynamics simulations paves the way to a deeper understanding of the role of internal dynamics and packing defects in the control of ligand binding in heme proteins. 相似文献
84.
85.
Tonachini L Monticone M Puri C Tacchetti C Pinton P Rizzuto R Cancedda R Tavella S Castagnola P 《Journal of cellular physiology》2004,201(3):470-482
We have recently identified a chondrocyte protein with a poly-proline region, referred to as CHPPR, and showed that this protein is expressed intracellularly in chick embryo chondrocytes. Conventional fluorescence and confocal localization of CHPPR shows that CHPPR is sorted to mitochondria. Furthermore, immunoelectron microscopy of CHPPR transfected cells demonstrates that this protein is mostly associated with the mitochondrial inner membranes. Careful analysis of CHPPR expressing cells reveals, instead of the regular mitochondrial tubular network, the presence of a number of small spheroid mitochondria. Here we show that the domain responsible for network-spheroid transition spans amino acid residues 182-309 including the poly-proline region. Functional analyses of mitochondrial activity rule out the possibility of mitochondrial damage in CHPPR transfected cells. Since cartilage expresses high levels of CHPPR mRNA when compared to other tissues and because CHPPR is associated with late stages of chondrocyte differentiation, we have investigated mitochondrial morphology in hypertrophic chondrocytes by MitoTracker Orange labeling. Confocal microscopy shows that these cells have spheroid mitochondria. Our data demonstrate that CHPPR is able to promote mitochondrial fission with a sequence specific mechanism suggesting that this event may be relevant to late stage of chondrocyte differentiation. 相似文献
86.
87.
Gian-Pietro Di Sansebastiano Massimiliano Gigante Stefania De Domenico Gabriella Piro Giuseppe Dalessandro 《Plant signaling & behavior》2006,1(2):77-85
Exocytosis molecular mechanisms in plant cells are not fully understood. The full characterization of molecular determinants, such as SNAREs, for the specificity in vesicles delivery to the plasma membrane should shed some light on these mechanisms. Nicotiana tabacum Syntaxin 1 (NtSyr1 or SYP121) is a SNARE protein required for ABA control of ion channels and appears involved in the exocytosis of exogenous markers.NtSyr1 is mainly localized on the plasma membrane, but when over expressed the protein also appears on endomembranes. Since NtSyr1 is a tail-anchored protein inserted into the target membrane post-translationally, it is not clear whether its initial anchoring site is the ER or the plasma membrane.In this study, we investigated the sorting events of NtSyr1 in vivo using its full-length cDNA or its C-terminal domain, fused to a GFP tag and transiently expressed in protoplasts or in the leaves of Nicotiana tabacum cv. SR1. Five chimeras were produced of which two were useful to investigate the protein sorting within the endomembrane system. One (GFP-H3M) had a dominant negative effect on exocytosis; the other one (SP1-GFP) resulted in a slow targeting to the same localization of the full-length chimera (GFP-SP1). The insertion of signal peptides on SP1-GFP further characterized the insertion site for this protein. Our data indicates that NtSyr1 is firstly anchored on ER membrane and then sorted to plasma membrane.Key Words: syntaxins, SNAREs, GFP tagging, exocytosis, secretion, protoplasts, dominant negative mutant 相似文献
88.
Centaurium pulchellum is an annual herb native to Europe, but introduced in South America, where it is widely used in the preparation of digestive infusions and bitter drinks. In this species, a wide variation in the aperture pattern of pollen grains was reported and has been attributed to environmental factors or irregularities at meiosis. For this reason, cytological and palynological studies have been undertaken on two different populations. The pollen grain analysis showed that some types are more frequent within each population, but the most common forms were the typical 3-colporate and 4-colporate. The cytological analysis revealed that the analyzed populations of C. pulchellum have chromosome number 2n = 36. The presence of tetravalents at meiosis strongly suggests that these populations are autotetraploid based on x = 9. The meiotic behavior showed a significant percentage of irregularities in different phases: off-plate bivalents, precocious segregation, laggard chromosomes, bridges, and cytomixis. However these irregularities are not related to the variation in the aperture pattern of pollen grains. The heteromorphism in the pollen grains observed in C. pulchellum could be a normal condition to which the species is well adapted. 相似文献
89.
Marco Paoloni Massimiliano Mangone Giancarlo Fratocchi Massimiliano Murgia Vincenzo Maria Saraceni Valter Santilli 《Journal of biomechanics》2010,43(9):1794-1798
Patients with patellofemoral pain syndrome (PFPS) often report discomfort and pain during walking. To date, most of the studies conducted to determine gait alterations in PFPS patients have focused on sagittal plane alterations. Physiological and biomechanical factors, however, suggest that frontal and transverse plane alterations may be involved in PFPS. We therefore decided to conduct a kinematic and kinetic evaluation on all three planes in 9 PFPS subjects and 9 healthy sex- and age-matched controls. General gait characteristics were similar in patients and controls, with the exception of swing velocity, which was lower in PFPS patients. Patients also displayed an increased knee abductor and external rotator moments in loading response, and reduced knee extensor moment both in loading response and in terminal stance. We speculate that these findings may be linked both to a pain-avoiding gait pattern and to alterations in the timing of activation of different components of the quadriceps muscle, which is typical of PFPS. The relevance for clinicians is this gait pattern may represent a biomechanical risk factor for future knee osteoarthritis. We therefore recommend that treatments aimed at PFPS should also attempt to restore a correct walking pattern. 相似文献
90.
Gianluca Storci Pasquale Sansone Sara Mari Gabriele D'Uva Simona Tavolari Tiziana Guarnieri Mario Taffurelli Claudio Ceccarelli Donatella Santini Pasquale Chieco Kenneth B. Marcu Massimiliano Bonafè 《Journal of cellular physiology》2010,225(3):682-691
Extracellular and intracellular mediators of inflammation, such as tumor necrosis factor alpha (TNFα) and NF‐kappaB (NF‐κB), play major roles in breast cancer pathogenesis, progression and relapse. SLUG, a mediator of the epithelial–mesenchymal transition process, is over‐expressed in CD44+/CD24? tumor initiating breast cancer cells and in basal‐like carcinoma, a subtype of aggressive breast cancer endowed with a stem cell‐like gene expression profile. Cancer stem cells also over‐express members of the pro‐inflammatory NF‐κB network, but their functional relationship with SLUG expression in breast cancer cells remains unclear. Here, we show that TNFα treatment of human breast cancer cells up‐regulates SLUG with a dependency on canonical NF‐κB/HIF1α signaling, which is strongly enhanced by p53 inactivation. Moreover, SLUG up‐regulation engenders breast cancer cells with stem cell‐like properties including enhanced expression of CD44 and Jagged‐1 in conjunction with estrogen receptor alpha down‐regulation, growth as mammospheres, and extracellular matrix invasiveness. Our results reveal a molecular mechanism whereby TNFα, a major pro‐inflammatory cytokine, imparts breast cancer cells with stem cell‐like features, which are connected to increased tumor aggressiveness. J. Cell. Physiol. 225: 682–691, 2010. © 2010 Wiley‐Liss, Inc. 相似文献