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41.
Impaired biosynthetic processing of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, constitutes the most common cause of CF. Recently, we have identified a distinct category of mutation, caused by premature stop codons and frameshift mutations, which manifests in diminished expression of COOH-terminally truncated CFTR at the cell surface. Although the biosynthetic processing and plasma membrane targeting of truncated CFTRs are preserved, the turnover of the complex-glycosylated mutant is sixfold faster than its wild-type (wt) counterpart. Destabilization of the truncated CFTR coincides with its enhanced susceptibility to proteasome-dependent degradation from post-Golgi compartments globally, and the plasma membrane specifically, determined by pulse-chase analysis in conjunction with cell surface biotinylation. Proteolytic cleavage of the full-length complex-glycosylated wt and degradation intermediates derived from both T70 and wt CFTR requires endolysosomal proteases. The enhanced protease sensitivity in vitro and the decreased thermostability of the complex-glycosylated T70 CFTR in vivo suggest that structural destabilization may account for the increased proteasome susceptibility and the short residence time at the cell surface. These in turn are responsible, at least in part, for the phenotypic manifestation of CF. We propose that the proteasome-ubiquitin pathway may be involved in the peripheral quality control of other, partially unfolded membrane proteins as well. 相似文献
42.
Tilted peptides are short sequence fragments (10-20 residues long) that possess an asymmetric hydrophobicity gradient along their sequence when they are helical. Due to this gradient, they adopt a tilted orientation towards a single lipid/water interface and destabilize the lipids. We have detected those peptides in many different proteins with various functions. While being all tilted-oriented at a single lipid/water interface, no consensus sequence can be evidenced. In order to better understand the relationships between their lipid-destabilizing activity and their properties, we used IMPALA to classify the tilted peptides. This method allows the study of interactions between a peptide and a modeled lipid bilayer using simple restraint functions designed to mimic some of the membrane properties. We predict that tilted peptides have access to a wide conformational space in membranes, in contrast to transmembrane and amphipathic helices. In agreement with previous studies, we suggest that those metastable configurations could lead to the perturbation of the acyl chains organization and could be a general mechanism for lipid destabilization. Our results further suggest that tilted peptides fall into two classes: those from proteins acting on membrane behave differently than destabilizing fragments from interfacial proteins. While the former have equal access to the two layers of the membrane, the latter are confined within a single lipid layer. This could be in relation with the organization of lipid substrate on which the peptides physiologically act. 相似文献
43.
González-Montalbán N Villaverde A Aris A 《Biochemical and biophysical research communications》2007,355(3):637-642
The aggregation of proteins in the form of amyloid fibrils and plaques is the characteristic feature of some pathological conditions ranging from neurodegenerative disorders to systemic amyloidoses. The mechanisms by which the aggregation processes result in cell damage are under intense investigation but recent data indicate that prefibrillar aggregates are the most proximate mediators of toxicity rather than mature fibrils. Since it has been shown that prefibrillar forms of the nondisease-related misfolded proteins are highly toxic to cultured mammalian cells we have studied the cytoxicity associated to bacterial inclusion bodies that have been recently described as protein deposits presenting amyloid-like structures. We have proved that bacterial inclusion bodies composed by a misfolding-prone beta-galactosidase fusion protein are clearly toxic for mammalian cells but the beta-galactosidase wild type enzyme forming more structured thermal aggregates does not impair cell viability, despite it also binds and enter into the cells. These results are in the line that the most cytotoxic aggregates are early prefibrilar assemblies but discard the hypothesis that the membrane destabilization is the key event to subsequent disruption of cellular processes, such as ion balance, oxidative state and the eventually cell death. 相似文献
44.
Aristeidis Giannakopoulos Eleana F. Stavrou Nicholas Zoumbos Aglaia Athanassiadou 《Journal of molecular biology》2009,387(5):1239-20
The scaffold/matrix attachment regions (S/MARs) are chromosomal elements that participate in the formation of chromatin domains and have origin of replication support functions. Because of all these functions, in recent years, they have been used as part of episomal vectors for gene transfer. The S/MAR of the human β-interferon gene has been shown to support efficient episome retention and transgene expression in various mammalian cells. In Jurkat and other cells, DNA plasmid vectors containing Epstein-Barr virus origin of replication (EBV OriP) and the EBV nuclear antigen-1 gene mediate prolonged episome retention in the host cell nucleus, which, however, diminishes over time. In order to enhance retention, we combined this system with an S/MAR element. Unexpectedly, this completely eliminated the capacity of episomes to replicate. Calculation of the stress-induced DNA duplex destabilization profile of the vectors suggested that the S/MAR element had created an increase in molecular stability at the OriP site that may have disturbed replicative potential. In contrast, introduction of an alternative initiation of replication region from the β-globin gene locus, instead of EBV OriP and the EBV nuclear antigen-1 gene, restored replicative capacity and enhanced episome retention mediated by the S/MAR. These effects were associated with a destabilization profile at the initiation of replication region. These data demonstrate a correlation between S/MAR-mediated vector retention and the presence of an unstable duplex at a replication origin, in this particular setting. We consider that the calculation of stress-induced duplex destabilization may be an informative first step in the design of units that replicate extrachromosomally, particularly as the latter present a safer and, therefore, attractive alternative to integrating viral vectors for gene therapy applications. 相似文献
45.
Shirin Jamshidi 《Journal of biomolecular structure & dynamics》2013,31(2):404-417
Catalytic mechanism of orotidine 5′-monophosphate decarboxylase (OMPDC), one of the nature most proficient enzymes which provides large rate enhancement, has not been fully understood yet. A series of 30?ns molecular dynamics (MD) simulations were run on X-ray structure of the OMPDC from Saccharomyces cerevisiae in its free form as well as in complex with different ligands, namely 1-(5′-phospho-D-ribofuranosyl) barbituric acid (BMP), orotidine 5′-monophosphate (OMP), and 6-phosphonouridine 5′-monophosphate (PMP). The importance of this biological system is justified both by its high rate enhancement and its potential use as a target in chemotherapy. This work focuses on comparing two physicochemical states of the enzyme (protonated and deprotonated Asp91) and three ligands (substrate OMP, inhibitor, and transition state analog BMP and substrate analog PMP). Detailed analysis of the active site geometry and its interactions is properly put in context by extensive comparison with relevant experimental works. Our overall results show that in terms of hydrogen bond occupancy, electrostatic interactions, dihedral angles, active site configuration, and movement of loops, notable differences among different complexes are observed. Comparison of the results obtained from these simulations provides some detailed structural data for the complexes, the enzyme, and the ligands, as well as useful insights into the inhibition mechanism of the OMPDC enzyme. Furthermore, these simulations are applied to clarify the ambiguous mechanism of the OMPDC enzyme, and imply that the substrate destabilization and transition state stabilization contribute to the mechanism of action of the most proficient enzyme, OMPDC. 相似文献
46.
The interactions that lead to structure transitions in oil-in-water emulsions were investigated using high-resolution ultrasonic
spectroscopy. High methoxyl pectin (HMP) was added to the emulsions at various concentrations and the dynamics of aggregation
induced by changes in pH were observed. Two independent ultrasonic parameters, velocity and attenuation, were measured as
a function of time or pH. At pH 6.8, both velocity and attenuation of sound changed as a function of HMP concentration. During
acidification, caused by the addition of glucono-δ-lactone, there were small changes in the overall ultrasonic velocity, but
it was possible to relate these changes to the structural changes in the emulsion. The values of ultrasonic attenuation decreased
at high pH with increasing amount of HMP, indicating changes in the flocculation state of the oil droplets caused by depletion
forces. During acidification at pH 5.4, emulsions containing HMP showed a steep increase in the ultrasonic attenuation, and
this pH corresponds to the pH of association of HMP with the casein-covered oil droplets. The adsorption of HMP onto the interface
causes a rearrangement of the oil droplets, and the emulsions containing sufficient amounts of HMP no longer gel at acid pH.
This is well described by the ultrasonic attenuation changes in the various emulsions. This research demonstrated for the
first time that ultrasonic spectroscopy can be employed for in situ monitoring and analysis of acid-induced destabilization
of food emulsions. 相似文献
47.
48.
Kiselyova OI Yaminsky IV Karpova OV Rodionova NP Kozlovsky SV Arkhipenko MV Atabekov JG 《Journal of molecular biology》2003,332(2):321-325
Recently we have reported that a selective binding of potato virus X (PVX)-coded movement protein (termed TGBp1 MP) to one end of a polar coat protein (CP) helix converted viral RNA into a translatable form and induced a linear destabilization of the whole helical particle. Here, the native PVX virions, RNase-treated (PVX(RNA-DEG)) helical particles lacking intact RNA and their complexes with TGBp1 (TGBp1-PVX and TGBp1-PVX(RNA-DEG)), were examined by atomic force microscopy (AFM). When complexes of the TGBp1 MP with PVX were examined by means of AFM in liquid, no structural reorganization of PVX particles was observed. By contrast, the products of TGBp1-dependent PVX degradation termed "beads-on-string" were formed under conditions of AFM in air. The AFM images of PVX(RNA-DEG) were indistinguishable from images of native PVX particles; however, the TGBp1-dependent disassembly of the CP-helix was triggered when the TGBp1-PVX(RNA-DEG) complexes were examined by AFM, regardless of the conditions used (in air or in liquid). Our data supported the idea that binding of TGBp1 to one end of the PVX CP-helix induced linear destabilization of the whole helical particle, which may lead to its disassembly under conditions of AFM. 相似文献
49.
Regulation of cell elongation is important for plant morphogenesis. Many studies have shown that cortical microtubules play crucial roles during cell elongation and that microtubule stability, organization, and dynamics are regulated by microtubule regulatory proteins.1 Recently, we reported that a novel protein from Arabidopsis, termed microtubule-destabilizing protein 25 (MDP25), functions as a negative regulator of hypocotyl cell elongation. MDP25 destabilizes microtubules and exerts its effect on microtubules as a result of transient elevation of cytosolic calcium levels.2 相似文献
50.
Destabilization of medial meniscus (DMM) model is an important tool for studying the pathophysiological roles of numerous arthritis associated molecules in the pathogenesis of osteoarthritis (OA) in vivo. However, the detailed, especially the visualized protocol for establishing this complicated model in mice, is not available. Herein we took advantage of wildtype and progranulin (PGRN)-/- mice as examples to introduce a protocol for inducing DMM model in mice, and compared the onset of OA following establishment of this surgically induced model. The operations performed on mice were either sham operation, which just opened joint capsule, or DMM operation, which cut the menisco-tibial ligament and caused destabilization of medial meniscus. Osteoarthritis severity was evaluated using histological assay (e.g. Safranin O staining), expressions of OA-associated genes, degradation of cartilage extracellular matrix molecules, and osteophyte formation. DMM operation successfully induced OA initiation and progression in both wildtype and PGRN-/- mice, and loss of PGNR growth factor led to a more severe OA phenotype in this surgically induced model. 相似文献