共查询到20条相似文献,搜索用时 15 毫秒
1.
Previous reports from this laboratory have demonstrated that lamin B is reversibly methylesterified in a cell cycle-dependent manner. The site of this methylation, however, was not identified. In this report, we describe a single major methylated product obtained following reversed-phase high-performance liquid chromatographic analysis of peptides generated by proteolytic digestion of lamin B from rat liver nuclear envelopes. This peptide was retained on a lamin B COOH-terminal-specific antibody-affinity column, and COOH-terminal localization was confirmed by amino acid sequencing. Two other COOH-terminal peptides were found but were not methylated and differed in sequence by at least a single residue from the methylated peptide, indicating the existence of two lamin B gene products. Tetrapeptides, representing the putative mature COOH termini of lamin B, K-ras-2A, and unprocessed lamin A, were synthesized with or without farnesyl modification of the COOH-terminal cysteines. All three farnesylated peptides served as substrates for the partially purified lamin B methyltransferase with apparent Km values of 4.5, 0.69, and 21 microM, respectively. Nonfarnesylated peptides were not substrates for the enzyme. The three farnesylated peptides were also effective to varying degrees at inhibiting the methylation of lamin B and other cellular proteins in cell lysates. 相似文献
2.
Internalization of fluorescently labeled CPPs, pVEC, transportan and scrambled pVEC, in a range of plant cells was investigated. Cellular uptake of the peptides was found to be tissue dependent. pVEC and transportan were distinctly internalized in triticale mesophyll protoplasts, onion epidermal cells, leaf bases and root tips of seven-day old triticale seedlings but showed negligible florescence in coleoptile and leaf tips as observed under a fluorescence microscope. Further, pVEC and transportan uptake studies were focused on mesophyll protoplasts as a system of investigation. In fluorimetric studies transportan showed 2.3 times higher cellular internalization than pVEC in protoplasts, whereas scrambled pVEC failed to show any significant fluorescence. Effect of various factors on cellular internalization of pVEC and transportan in protoplasts was also investigated. The cellular uptake of both the peptides was concentration dependent and nonsaturable. The cellular uptake of pVEC and transportan was enhanced at low temperature (4 degrees C). The presence of endocytic/macropinocytosis inhibitors did not reduce the cellular uptake of the peptides, suggesting direct cell penetration, receptor-independent internalization of pVEC and transportan into the plant cells. 相似文献
3.
Yokoyama F Suzuki N Kadoya Y Utani A Nakatsuka H Nishi N Haruki M Kleinman HK Nomizu M 《Biochemistry》2005,44(28):9581-9589
Laminin alpha chains show diverse biological functions in a chain-specific fashion. The laminin G-like modules (LG modules) of the laminin alpha chains consist of a 14-stranded beta-sheet sandwich structure with biologically active sequences found in the connecting loops. Previously, we reported that connecting loop regions between beta-strands E and F in the mouse laminin alpha chain LG4 modules exhibited chain-specific activities. In this study, we focus on the homologous loop regions in human laminin alpha chain LG4 modules using five synthetic peptides (hEF-1-hEF-5). These homologous peptides induced chain-specific cellular responses in various cell types. Next, to examine the dual-receptor recognition model, we synthesized chimeras (cEF13A-cEF13E) derived from peptides hEF-1 and hEF-3. All of the chimeric peptides promoted fibroblast attachment as well as the parental peptides. Attachment of fibroblasts to cEF13A and cEF13B was inhibited by anti-integrin alpha2 and beta1 antibodies and by heparin, while cell adhesion to cEF13C, cEF13D, and cEF13E was blocked only by heparin. Actin organization of fibroblasts on cEF13C was not different from that on hEF-3, but cEF13B induced membrane ruffling at the tips of the actin stress fibers. These results suggest that cEF13B had bifunctional effects on cellular behaviors through alpha2beta1 integrin and heparin/heparan sulfate proteoglycan. We conclude that the approach utilizing chimeric peptides is useful for examining cellular mechanisms in dual-receptor systems. 相似文献
4.
Thorén PE Persson D Isakson P Goksör M Onfelt A Nordén B 《Biochemical and biophysical research communications》2003,307(1):100-107
Cell-penetrating peptides are regarded as promising vectors for intracellular delivery of large, hydrophilic molecules, but their mechanism of uptake is poorly understood. Since it has now been demonstrated that the use of cell fixation leads to artifacts in microscopy studies on the cellular uptake of such peptides, much of what has been considered as established facts must be reinvestigated using live (unfixed) cells. In this work, the uptake of analogs of penetratin, Tat(48-60), and heptaarginine in two different cell lines was studied by confocal laser scanning microscopy. For penetratin, an apparently endocytotic uptake was observed, in disagreement with previous studies on fixed cells found in the literature. Substitution of the two tryptophan residues, earlier reported to be essential for cellular uptake, did not alter the uptake characteristics. A heptaarginine peptide, with a tryptophan residue added in the C-terminus, was found to be internalized by cells via an energy-independent, non-endocytotic pathway. Finally, a crucial role for arginine residues in penetratin and Tat(48-60) was demonstrated. 相似文献
5.
Peptides have been proposed to function in intracellular signaling within the cytosol. Although cytosolic peptides are considered to be highly unstable, a large number of peptides have been detected in mouse brain and other biological samples. In the present study, we evaluated the peptidome of three diverse cell lines: SH-SY5Y, MCF7, and HEK293 cells. A comparison of the peptidomes revealed considerable overlap in the identity of the peptides found in each cell line. The majority of the observed peptides are not derived from the most abundant or least stable proteins in the cell, and approximately half of the cellular peptides correspond to the N- or C- termini of the precursor proteins. Cleavage site analysis revealed a preference for hydrophobic residues in the P1 position. Quantitative peptidomic analysis indicated that the levels of most cellular peptides are not altered in response to elevated intracellular calcium, suggesting that calpain is not responsible for their production. The similarity of the peptidomes of the three cell lines and the lack of correlation with the predicted cellular degradome implies the selective formation or retention of these peptides, consistent with the hypothesis that they are functional in the cells. 相似文献
6.
Multivalency represents a critical parameter in cell biology responsible for the overall avidity of low-affinity interactions and the triggering of cellular events. Functions such as catalytic activity, cellular uptake, or localization are frequently linked to the oligomeric state of a protein. This study explores the impact of multivalency on the import and routing of peptides into cells. Specifically, cationic import sequences such as decaarginine, decalysine, and the HIV Tat peptide (GRKKRRQRRRAP, residues 48-59) as well as the nuclear localization sequence from SV40 large T-antigen were assembled into defined peptide oligomers by fusing them to the tetramerization domain of human p53 (residues 325-355, hp53(tet) domain). The resulting tetravalent peptides typically displayed between 10- and 100-fold enhancements in cellular import and intracellular routing properties in relation to their monomeric homologues. These peptides were not toxic to cells. Flow cytometry results and transfection assays indicated that tetravalent decaarginyl peptides (10R-p53(tet) and NLS-10R-p53(tet)) were the peptides most efficiently routed into cells. Their mechanism of import was subsequently examined on unfixed, viable cells using a combination of metabolic inhibitors, flow cytometry, and microscopy techniques. These studies revealed that tetravalent arginine-rich peptides bind to heparan sulfate on the cell surface, are internalized at 37 degrees C, but not at 4 degrees C, via a clathrin-mediated pathway, and accumulate into endosome-like acidic compartments. A fraction of these tetravalent peptides access the cytosol and accumulate in the nucleus of cells. This study concludes that the oligomerization of proteins harboring arginine-rich peptide chains may profoundly influence their ability to enter and be routed into cells. 相似文献
7.
Studies on the cellular uptake of substance P and lysine-rich, KLA-derived model peptides 总被引:4,自引:0,他引:4
In the last decade many peptides have been shown to be internalized into various cell types by different, poorly characterized mechanisms. This review focuses on uptake studies with substance P (SP) aimed at unravelling the mechanism of peptide-induced mast cell degranulation, and on the characterization of the cellular uptake of designed KLA-derived model peptides.Studies on structure-activity relationships and receptor autoradiography failed to detect specific peptide receptors for the undecapeptide SP on mast cells. In view of these findings, a direct interaction of cationic peptides with heterotrimeric G proteins without the participation of a receptor has been proposed. Such a process would require insertion into and translocation of peptides across the plasma membrane.In order to clarify whether a transport of cationic peptides into rat peritoneal mast cells is possible, transport studies were performed by confocal laser scanning microscopy (CLSM) using fluorescence-labeled Arg(3),Orn(7)-SP and its D-amino acid analog, all-D-Arg(3),Orn(7)-SP, as well as by electron microscopic autoradiography using (3)H-labelled SP and (125)I-labelled all-D-SP. The results obtained by CLSM directly showed translocation of SP peptides into pertussis toxin-treated cells. Kinetic experiments indicated that the translocation process was rapid, occurring within a few seconds. Mast cell degranulation induced by analog of magainin 2 amide, neuropeptide Y and the model peptide acetyl-KLALKLALKALKAALKLA-amide was also found to be very fast, pointing to an extensive translocation of the peptides. In order to learn more about structural requirements for the cellular uptake of peptides, the translocation behavior of a set of systematically modified KLA-based model peptides has been studied in detail. By two different protocols for determining the amount of internalized peptide, evidence was found that the structure of the peptides only marginally affects their uptake, whereas the efflux of cationic, amphipathic peptides is strikingly diminished, thus allowing their enrichment within the cells. Although the mechanism of cellular uptake, consisting of energy-dependent and -independent contributions, is not well understood, KLA-derived peptides have been shown to deliver various cargos (PNAs, peptides) into cells. The results obtained with SP- and KLA-derived peptides are discussed in the context of the current literature. 相似文献
8.
S Kohan B Fr?ysa E Cederlund T Fairwell R Lerner J Johansson S Khan M Ritzen H J?rnvall S Cekan 《FEBS letters》1986,199(2):242-248
A 94-residue polypeptide isolated from human seminal plasma and its chemically synthesized C-terminal 28-residue segment were studied in an in vitro inhibin bioassay utilizing rat pituitary cell cultures. Both peptides have previously been claimed to have inhibin activities, and the effects on the secretion and cellular content of gonadotrophins (FSH and LH) were now assessed in the in vitro assay. No inhibition was found. After 72 h of culture, both the cellular content and the spontaneous as well as the LHRH-stimulated release of bioactive or immunoactive FSH and LH remained unaffected. Similarly, no effects were found on the storage and/or release of prolactin, growth hormone, or thyrotropin. We conclude that both the native 94-residue peptide and the synthetic replicate of its C-terminal 28-residue segment, do not influence the pituitary FSH secretion when assessed in this in vitro system. 相似文献
9.
Jorunn Stamnaes Siri Dorum Burkhard Fleckenstein Daniel Aeschlimann Ludvig M. Sollid 《Amino acids》2010,39(5):1183-1191
Transglutaminase 2 (TG2) is well characterized as the main autoantigen of celiac disease. The ability of TG2 to deamidate
and crosslink gluten peptides is essential for the gluten-dependent production of TG2 specific autoantibodies. In patients
with primarily extraintestinal manifestation of gluten sensitivity the repertoire of autoantibodies may be different. In dermatitis
herpetiformis (DH), TG3 appears to be the target autoantigen whereas in gluten ataxia (GA) autoantibodies reactive with TG6
are present. A functional role for TG3 and TG6 in these diseases has yet to be described. It is also not known whether these
enzymes can use gluten peptides implicated in the pathology as substrates. We here report that similar to TG2, TG3 and TG6
can specifically deamidate gluten T cell epitopes. However, the fine specificities of the enzymes were found to differ. TG2
can form covalent complexes with gluten by iso-peptide and thioester bonds. We found that both TG3 and TG6 were able to complex
with gluten peptides through thioester linkage although less efficiently than TG2, whereas TG6 but not TG3 was able to form
iso-peptide linked complexes. Our findings lend credence to the notion that TG3 and TG6 are involved in the gluten-induced
autoimmune responses of DH and GA. 相似文献
10.
家蝇抗菌肽的抑菌动力学研究及其机理初探 总被引:6,自引:0,他引:6
利用鼠伤寒沙门氏菌针刺诱导家蝇幼虫表达抗菌肽,对抗菌肽的抑菌动力学进行研究,并通过抗菌肽样品对不同细菌动力学特性的研究出发对抗菌肽抑菌作用机制进行探讨。研究发现抗菌肽样品的活性与作用时间有关,24h内出现一到两个活性峰,同一抗菌肽样品对不同细菌的抑菌动力学有差异,抗菌肽的抑菌动力学机制应该与它的的抑菌作用机制有关。通过电镜观测、细胞磷代谢、紫外吸收物测定以及抗菌肽与细菌DNA相互作用结果可知,微生物诱导家蝇表达的抗菌肽样品不仅能够造成细菌细胞的快速坍塌破裂而且能够破坏细胞核心,与DNA结合作用。抗菌肽抑菌动力学的解释:微生物诱导产物中含有两类抗菌肽,一类抗菌肽能造成细胞膜的快速坍塌破裂形成第一个活性峰;另一类抗菌肽可进入细胞,破坏细胞核心,造成紫外吸收物大量外泄形成第二个活性峰。 相似文献
11.
12.
ProSAAS is the precursor for some of the most abundant peptides found in mouse brain and other tissues, including peptides named SAAS, PEN, and LEN. Both SAAS and LEN are found in big and little forms due to differential processing. Initial processing of proSAAS is mediated by furin (and/or furin-like enzymes) and carboxypeptidase D, while the smaller forms are generated by secretory granule prohormone convertases and carboxypeptidase E. In mouse hypothalamus, PEN and big LEN colocalize with neuropeptide Y. In the present study, little LEN and SAAS were detected in mouse hypothalamus but not in cell bodies of neuropeptide Y-expressing neurons. PEN and big LEN show substantial colocalization in hypothalamus, but big LEN and little LEN do not. An antiserum to SAAS that detects both big and little forms of this peptide did not show substantial colocalization with PEN or big LEN. To further study this, the AtT-20 cells mouse pituitary corticotrophic cell line was transfected with rat proSAAS and the distribution of peptides examined. As found in mouse hypothalamus, only some of the proSAAS-derived peptides colocalized with each other in AtT-20 cells. The two sites within proSAAS that are known to be efficiently cleaved by furin were altered by site-directed mutagenesis to convert the P4 Arg into Lys; this change converts the sequences from furin consensus sites into prohormone convertase consensus sites. Upon expression of the mutated form of proSAAS in AtT-20 cells, there was significantly more colocalization of proSAAS-derived peptides PEN and SAAS. Taken together, these results indicate that proSAAS is initially cleaved in the Golgi or trans-Golgi network by furin and/or furin-like enzymes and the resulting fragments are sorted into distinct vesicles and further processed by additional enzymes into the mature peptides. 相似文献
13.
Bhutani N Piccirillo R Hourez R Venkatraman P Goldberg AL 《The Journal of biological chemistry》2012,287(21):17471-17482
In neurodegenerative diseases caused by extended polyglutamine (polyQ) sequences in proteins, aggregation-prone polyQ proteins accumulate in intraneuronal inclusions. PolyQ proteins can be degraded by lysosomes or proteasomes. Proteasomes are unable to hydrolyze polyQ repeat sequences, and during breakdown of polyQ proteins, they release polyQ repeat fragments for degradation by other cellular enzymes. This study was undertaken to identify the responsible proteases. Lysosomal extracts (unlike cytosolic enzymes) were found to rapidly hydrolyze polyQ sequences in peptides, proteins, or insoluble aggregates. Using specific inhibitors against lysosomal proteases, enzyme-deficient extracts, and pure cathepsins, we identified cathepsins L and Z as the lysosomal cysteine proteases that digest polyQ proteins and peptides. RNAi for cathepsins L and Z in different cell lines and adult mouse muscles confirmed that they are critical in degrading polyQ proteins (expanded huntingtin exon 1) but not other types of aggregation-prone proteins (e.g. mutant SOD1). Therefore, the activities of these two lysosomal cysteine proteases are important in host defense against toxic accumulation of polyQ proteins. 相似文献
14.
Long stretches of glutamine (Q) residues are found in many cellular proteins. Expansion of these polyglutamine (polyQ) sequences is the underlying cause of several neurodegenerative diseases (e.g. Huntington's disease). Eukaryotic proteasomes have been found to digest polyQ sequences in proteins very slowly, or not at all, and to release such potentially toxic sequences for degradation by other peptidases. To identify these key peptidases, we investigated the degradation in cell extracts of model Q-rich fluorescent substrates and peptides containing 10-30 Q's. Their degradation at neutral pH was due to a single aminopeptidase, the puromycin-sensitive aminopeptidase (PSA, cytosol alanyl aminopeptidase). No other known cytosolic aminopeptidase or endopeptidase was found to digest these polyQ peptides. Although tripeptidyl peptidase II (TPPII) exhibited limited activity, studies with specific inhibitors, pure enzymes and extracts of cells treated with siRNA for TPPII or PSA showed PSA to be the rate-limiting activity against polyQ peptides up to 30 residues long. (PSA digests such Q sequences, shorter ones and typical (non-repeating) peptides at similar rates.) Thus, PSA, which is induced in neurons expressing mutant huntingtin, appears critical in preventing the accumulation of polyQ peptides in normal cells, and its activity may influence susceptibility to polyQ diseases. 相似文献
15.
We examined the effect of cellular metabolism of three alkyl-substituted amino acid ester phosphoramidate derivatives of stavudine in different cell lines. Marked cell-to-cell differences were found in both the rate of hydrolysis and chiral selectivity. This selectivity implies that different enzymes may be involved in the metabolism of these compounds depending on the cell type involved. Notably, both the methyl and ethyl substituted derivatives underwent hydrolysis in presence of various cell lines, whereas the tert-butyl substituted compound was resistant to hydrolysis implying that steric hindrance associated with this group along with electron density may play a key role in the hydrolysis profile of these compounds. Additionally we found this mimicked the hydrolysis profiles obtained for bacterial enzymes. Furthermore, our results suggest that the site of attack of the cellular enzymes is confined to the ester side chain of the molecule. This result is also consistent with our earlier observation using bacterial enzymes as well as using 'd' isomers. 相似文献
16.
The order of secretion of newly synthesized and older bioactive peptides was investigated using primary rat intermediate pituitary melanotropes, which synthesize, store, and secrete peptides derived from pro-ACTH/endorphin (PAE; also POMC). PAE-derived peptides produced by the cells were biosynthetically labeled by incubating the cells with radioactive amino acids at various times preceding the period during which secretion was examined; secreted and cellular peptides were characterized and quantitated by immunoprecipitation, using affinity-purified antibodies to selected regions of PAE, followed by polyacrylamide gel electrophoretic analysis. Release in the absence of secretagogues (basal or constitutive release) was compared to release in the presence of maximally effective levels of 8-bromo-cAMP and BaCl2 (stimulated or regulated release). Both cell types showed short-lived preferential basal release of newly synthesized and not fully mature peptides (less than 2-3 h old). Conversely, the cells showed preferential stimulated secretion of older peptides. A process of maturation occurred, taking 2-4 h, after which the secretion of newly synthesized and older peptides in response to secretagogues was nearly indistinguishable for the smallest product peptides. The data support a model of gradual processing of peptides from precursors into smaller products and maturation from molecules only available for basal release into peptides available for stimulated secretion as well as for basal release. Basal secretion was found to include mature peptides as well as intermediates and precursor molecules. The data do not support the existence of any preferential regulated secretion of newly synthesized peptides. 相似文献
17.
Spatial control over the distribution and the aggregation of arginine-glycine-aspartate (RGD) peptides at the nanoscale significantly affects cell responses. For example, nanoscale clustering of RGD peptides can induce integrins to cluster, thus triggering complete cell signaling. Dendrimers have a unique, highly branched, nearly spherical and symmetrical structure with low polydispersity, nanoscale size, and high functionality. Therefore, dendrimers are a class of ideal scaffold for construction of nanoscale dendritic RGD clusters in which RGD loading degree and cluster size can be finely adjusted. This new type of nanoscale dendritic RGD cluster will aid us to better understand the impact of spatial arrangement of RGD on cellular responses and to engineer RGD to trigger more favorable cellular responses. In this study, nanoscale dendritic RGD clusters were synthesized based on Starburst anionic G3.5 and cationic G4.0 polyamidoamine (PAMAM) dendrimers. The multiple terminal functional groups on the outermost layer of the dendrimer were coupled with RGD tripeptides. Biofunctionalized dendrimer structures were found to be highly dependent on the generation and the extent of peptide modification (ie, number of peptides per PAMAM dendrimer). Fluorescein isothiocyanate (FITC)-conjugated PAMAM dendrimers were utilized to monitor cellular internalization of dendrimers by adherent fibroblasts. Anionic G3.5-based dendritic RGD clusters have been shown to have no negative effect on fibroblast viability and a concentration-dependent effect on lowering cell adhesion on tissue culture polystyrene (TCPS) as that of free RGD. A similar concentration-dependent effect in cell viability and adhesion was also observed for cationic G4.0-based dendritic RGD clusters at lower but not at high concentrations. The results imply that the synthesized nanoscale dendritic RGD clusters have great potential for tissue engineering and drug delivery applications. 相似文献
18.
Effects of fibronectin-related peptides on cell spreading 总被引:6,自引:0,他引:6
Janet E. Silnutzer David W. Barnes 《In vitro cellular & developmental biology. Plant》1985,21(1):73-78
Summary Four synthetic peptides related in amino acid sequence to the area of the fibronectin molecule thought to be involved in cell
binding were tested for the ability to inhibit cell spreading promoted by several different attachment factors. Effects of
the peptides were assayed on a number of cell types. For all cell types tested, peptides effective at inhibiting fibronectin-promoted
cell spreading were found also to inhibit cell spreading promoted by human serum spreading factor (SF). These peptides were
also capable of inhibiting to a lesser extent cell spreading promoted by other attachment factors, although this phenomenon
was dependent on the cell type examined.
This work was supported by National Institutes of Health grant CA-40475
Editor's statement This paper relates cellular attachment mediated by dissimilar molecules. As such it offers potential insight
into common mechanisms of attachment. Gordon H. Sato 相似文献
19.
A Ito T Ogishima W Ou T Omura H Aoyagi S Lee H Mihara N Izumiya 《Journal of biochemistry》1985,98(6):1571-1582
One common and characteristic feature of the extension peptides of mitochondrial enzyme precursors is the presence of repeating short stretches of uncharged amino acids linked by basic amino acids. We synthesized several model peptides having this particular feature of the extension peptides. The peptides contained arginine or lysine as a basic amino acid residue linking sequences of two to four residues of leucine and alanine. We examined the effects of the peptides on the import of the precursors of two mitochondrial enzymes, cytochrome P-450(SCC) and adrenodoxin, and found that the peptides were generally inhibitory to the import of the precursors into mitochondria. The effective concentrations of some of the inhibitory peptides were as low as a few microM. The peptides containing lysine instead of arginine had an essentially similar inhibitory effect on the import. The peptides did not inhibit the binding of pre-P450(SCC) to the surface of mitochondria. The synthetic model peptides uncoupled oxidative phosphorylation of mitochondria prepared from either rat liver or bovine adrenal cortex, and induced leakage of enzymes from the inner compartments of mitochondria. However, the synthetic model peptides did not solubilize membrane-bound enzymes from mitochondria, suggesting that their effect on the membranes is different from that of detergents. The synthetic model peptides seem to bind to the membranes causing significant perturbation in the membrane structure, which is possibly related to the functions of the particular common sequence found in the extension peptides of mitochondrial enzyme precursors. 相似文献
20.
Reversible lipid attachment was investigated as a means to deliver small peptides into cells. Two labile straight chain alkyl motifs were developed: a cysteine dodecane disulfide (Cdd) building block and a tyrosine- or serine-myristate ester. Both moieties are cleaved on cell internalization and are compatible with Fmoc solid phase peptide synthesis. A series of fluorophore-labeled peptides that varied in lipophilic content, net charge, and charge distribution were synthesized. The peptides were screened for cellular uptake efficiency as monitored by fluorescence microscopy. Effective peptide transport is based on a distributed net positive charge introduced as lysine residues at the C and/or N terminus of the peptide and the presence of a hydrophobic domain exhibiting an estimated log P4.0. The incorporation of labile lipid motifs into peptides enhances lipophilic character of the peptides and contributes to cellular uptake with minimal alteration to the native sequence. 相似文献