共查询到20条相似文献,搜索用时 46 毫秒
1.
2.
3.
4.
5.
CL Luongo KA Dryden DL Farsetta RL Margraf TF Severson NH Olson BN Fields TS Baker ML Nibert 《Canadian Metallurgical Quarterly》1997,71(10):8035-8040
The 144-kDa lambda2 protein is a structural component of mammalian reovirus particles and contains the guanylyltransferase activity involved in adding 5' caps to reovirus mRNAs. After incubation of reovirus T3D core particles at 52 degrees C, the lambda2 protein became sensitive to partial protease degradation. Sequential treatments with heat and chymotrypsin caused degradation of a C-terminal portion of lambda2, leaving a 120K core-associated fragment. The four other proteins in cores--lambda1, lambda3, mu2, and sigma2--were not affected by the treatment. Purified cores with cleaved lambda2 were subjected to transmission cryoelectron microscopy and image reconstruction. Reconstruction analysis demonstrated that a distinctive outer region of lambda2 was missing from the modified cores. The degraded region of lambda2 corresponded to the one that contacts the base of the sigma1 protein fiber in reovirus virions and infectious subvirion particles, suggesting that the sigma1-binding region of lambda2 is near its C terminus. Cores with cleaved lambda2 were shown to retain all activities required to transcribe and cap reovirus mRNAs, indicating that the C-terminal region of lambda2 is dispensable for those functions. 相似文献
6.
7.
BACKGROUND: Replication of a plasmid derived from the Escherichia coli phage lambda initiates by binding of the lambda O protein initiator to the origin of lambda DNA replication, ori lambda. The lambda P protein participates in subsequent steps of assembly of the lambda replication complex. A function of lambda P required for replication complex assembly is inactivated at 43 degrees C by the ts1 mutation. RESULTS: We found that the lambda replication complex assembled at 30 degrees C survives the temperature upshift in lambda crotsPts1 plasmid-harbouring bacteria. We present several lines of evidence that in this system (in which the replication complex assembly does not occur), the replication complex assembled prior to the temperature upshift is inherited by one of two daughter plasmid copies at each replication round for more than 30 cell generations. The 'old' replication complex-driven replication is chloramphenicol-resistant and rifampicin-sensitive. This replication is dependent on lambda O and host dnaK, dnaJ and grpE chaperone gene functions. CONCLUSIONS: The lambda O-containing replication complex is inherited together with DNA and bears information how to initiate the next round of replication at ori lambda; thus, we consider that this phenomenon deserves to be called protein inheritance. 相似文献
8.
Lambda Xis, which is required for site-specific excision of phage lambda from the bacterial chromosome, has a much shorter functional half-life than Int, which is required for both integration and excision (R. A. Weisberg and M. E. Gottesman, p. 489-500, in A. D. Hershey, ed., The Bacteriophage Lambda, 1971). We found that Xis is degraded in vivo by two ATP-dependent proteases, Lon and FtsH (HflB). Xis was stabilized two- to threefold more than in the wild type in a lon mutant and as much as sixfold more in a lon ftsH double mutant at the nonpermissive temperature for the ftsH mutation. Integration of lambda into the bacterial chromosome was delayed in the lon ftsH background, suggesting that accumulation of Xis in vivo interferes with integration. Overexpression of Xis in wild-type cells from a multicopy plasmid inhibited integration of lambda and promoted curing of established lysogens, confirming that accumulation of Xis interferes with the ability of Int to establish and maintain an integrated prophage. 相似文献
9.
10.
J Opara M Szeliga-Cetnarska M Chromy J Dmytryk T Ickowicz 《Canadian Metallurgical Quarterly》1998,32(4):803-812
The Escherichia coli FtsH protein is a membrane-bound and ATP-dependent protease. In this study, we describe ATP-dependent conformational changes in FtsH as well as a polypeptide binding ability of this protein. A 33 kDa segment of FtsH became trypsin resistant in the presence of ATP. ATP and ATPgammaS prevented self-aggregation of detergent-solubilized FtsH-His6-Myc at 37 degrees C, again suggesting that the binding of ATP induces a conformational change in FtsH. Affinity chromatography showed that FtsH-His6-Myc can associate with denatured alkaline phosphatase (PhoA) but not with the native enzyme. Denatured PhoA also prevented the aggregation of FtsH, and these two proteins co-sedimented through a sucrose gradient. Binding between FtsH-His6-Myc and detergent-solubilized SecY was also demonstrated. Although FtsH-bound SecY was processed further for ATP-dependent proteolysis, FtsH-bound PhoA was not. Thus, FtsH association with denatured PhoA is uncoupled from proteolysis. Overproduction of FtsH significantly increased the cytoplasmic localization of the PhoA moiety of a MalF-PhoA hybrid protein, in which a charged residue had been introduced into a transmembrane segment. Thus, denatured PhoA binding of FtsH may also occur in vivo. 相似文献
11.
Although protein degradation is enhanced in muscle-wasting conditions and limits the rate of muscle growth in domestic animals, the proteolytic system responsible for degrading myofibrillar proteins in skeletal muscle is not well defined. The goals of this study were to evaluate the roles of the calpains (calcium-activated cysteine proteases) in mediating muscle protein degradation and the extent to which these proteases participate in protein turnover in muscle. Two strategies to regulate intracellular calpain activities were developed: overexpression of dominant-negative m-calpain and overexpression of calpastatin inhibitory domain. To express these constructs, L8 myoblast cell lines were transfected with LacSwitch plasmids, which allowed for isopropyl beta-D-thiogalactoside-dependent expression of the gene of interest. Inhibition of calpain stabilized fodrin, a well characterized calpain substrate. Under conditions of accelerated degradation (serum withdrawal), inhibition of m-calpain reduced protein degradation by 30%, whereas calpastatin inhibitory domain expression reduced degradation by 63%. Inhibition of calpain also stabilized nebulin. These observations indicate that calpains play key roles in the disassembly of sarcomeric proteins. Inhibition of calpain activity may have therapeutic value in treatment of muscle-wasting conditions and may enhance muscle growth in domestic animals. 相似文献
12.
Isolation and characterization of the phage T4 PinA protein, an inhibitor of the ATP-dependent lon protease of Escherichia coli 总被引:1,自引:0,他引:1
The bacteriophage T4 PinA protein, expression of which leads to inhibition of protein degradation in Escherichia coli cells, has been purified from cells carrying multiple copies of the pinA gene. PinA is a heat-stable protein with a subunit Mr of 18,800 and an isoelectric point of 4.6. Under nondenaturing conditions on a gel filtration column, PinA migrated in two peaks corresponding to a dimer and a tetramer. Purified PinA inhibited ATP-dependent protein degradation by Lon protease in vitro; it did not inhibit the activity of other E. coli ATP-dependent proteases, ClpAP or ClpYQ. Furthermore, PinA did not inhibit ATP-independent proteolysis in E. coli cell extracts. PinA binds with high affinity to Lon protease (Kd approximately 10 nM for dimer binding), and a complex with approximately 1 dimer of PinA per tetramer of Lon protease could be isolated by gel filtration. Lon activity was partially restored upon dilution of the PinA-Lon complex to subnanomolar concentrations, indicating that inhibition was reversible and that PinA did not covalently modify Lon protease. PinA was not cleaved by Lon protease, and heating the Lon-PinA complex at 65 degrees C denatured Lon protease and released active PinA. The properties of PinA in vitro suggest that PinA inhibits protein degradation in vivo by forming a tight, reversible complex with Lon protease. 相似文献
13.
14.
H Troeger 《Canadian Metallurgical Quarterly》1995,52(1):75-81
The key event associated with the initiation of angiogenesis is the localized degradation of the vascular basement membrane. Because of its complex structure, any remodelling and/or modification of the basement membrane must involve the co-ordinated function of a number of different enzyme systems. Type IV collagen is a major protein component (60-90%) of the basement membrane and its degradation is crucial to the initiation of angiogenesis. This study has focused on the mechanisms by which C6 astrocytoma cells degrade human type IV collagen. C6 astrocytoma cells use components of two major degradative pathways to degrade collagen type IV. The major matrix metalloproteinase identified is the activated form (68-KDa) of gelatinase A (72-KDa matrix metalloproteinase) and a serine sensitive 1000-KDa collagenase type IV degrading activity which appears to have the characteristics of a novel extracellular proteasome. 相似文献
15.
K Iizuka 《Canadian Metallurgical Quarterly》1994,69(1):112-119
The calcium-activated neutral protease, calpain, was tested for its proteolytic effects to degrade the cell membrane spectrin-like protein, fodrin, during hypoxia. Cardiac myocytes, isolated from neonatal rat hearts, were incubated under hypoxic conditions for 6 hours. The cell death during hypoxia rose to 80% after 6 hours. Extracellular protease activity was much more elevated during hypoxia, than in aerobic states at 6 hours. Intracellular protease activity was also elevated in hypoxia. These protease activities were markedly inhibited by the cysteine protease inhibitor E-64 and the calpain specific inhibitor, calpastatin. Hypoxic cell death was also suppressed by E-64. Cell membrane proteins prepared from hypoxic myocytes were examined with electroblots stained for fodrin by the peroxidase method. A 125 kilodalton immunoreactive degradation product of fodrin was found under hypoxic conditions. Treatment with E-64 inhibited both the appearance of this degradation band and the decrease in the content of fodrin. These observations indicate that calpain is activated during hypoxia and that it is related to cell membrane protein degradation, especially in fodrin. The data also suggest that protease inhibitor E-64 treatment may be beneficial in protection against hypoxic myocyte injury. 相似文献
16.
Bacteriophage lambda adsorbs to its Escherichia coli K12 host by interacting with a specific cell surface receptor, the outer membrane protein LamB. Previous genetic analyses led us to define a set of residues at the surface of LamB, which belong to the lambda receptor site. Further genetic studies indicated that the C-terminal portion of J, the tail fibre protein of lambda, was directly involved in the recognition of the receptor site. The present work describe first in vitro studies on the interactions between J and LamB. The J gene of lambda was cloned into a plasmid vector under ptac promoter control and expressed in E. coli. We showed that J could be expressed at high levels (up to 28% of whole cell proteins), in an insoluble form. Anti-J antibodies, induced in rabbits immunized with insoluble J extracts, appeared to specifically neutralize lambda infection. Under defined conditions of extraction, the J protein was obtained in a soluble form. We showed that solubilized J was able to interact with LamB trimers in vitro. Implications for future studies on the interactions between LamB and J are discussed. 相似文献
17.
18.
19.
The molecular chaperone ClpX of Escherichia coli plays two distinct functions for bacteriophage Mu DNA replication by transposition. As specificity component of a chaperone-linked protease, it recognizes the Mu immunity repressor for degradation by the peptidase component ClpP, thus derepressing Mu transposition functions. After strand exchange has been promoted by MuA transposase, ClpX alone can alter the conformation of the transpososome (the complex of MuA with Mu ends), and the remodeled MuA promotes transition to replisome assembly. Although ClpXP can degrade MuA, the presence of both ClpP and ClpX in the reconstituted transposition system did not destroy MuA essential for initiation of DNA replication by specific host replication enzymes. Levels of ClpXP needed to overcome inhibition by the repressor did not prevent MuA from promoting strand transfer, and ClpP stimulated alteration of the transpososome by ClpX. Apparently intact MuA was still present in the resulting transpososome, promoting initiation of Mu DNA replication by specific replication enzymes. The results indicate that ClpXP can discriminate repressor and MuA in the transpososome as substrates of the protease or the molecular chaperone alone, degrading repressor while remodeling MuA for its next critical function. 相似文献
20.
It was previously demonstrated that while lysogenic development of bacteriophage lambda in Escherichia coli proceeds normally at low temperature (20-25 degrees C), lytic development is blocked under these conditions owing to the increased stability of the phage CII protein. This effect was proposed to be responsible for the increased stimulation of the pE promoter, which interferes with expression of the replication genes, leading to inhibition of phage DNA synthesis. Here we demonstrate that the burst size of phage lambda cIb2, which is incapable of lysogenic development, increases gradually over the temperature range from 20 to 37 degrees C, while no phage progeny are observed at 20 degrees C. Contrary to previous reports, it is possible to demonstrate that pE promoter activation by CII may be more efficient at lower temperature. Using density-shift experiments, we found that phage DNA replication is completely blocked at 20 degrees C. Phage growth was also inhibited in cells overexpressing cII, which confirms that CII is responsible for inhibition of phage DNA replication. Unexpectedly, we found that replication of plasmids derived from bacteriophage lambda is neither inhibited at 20 degrees C nor in cells overexpressing cII. We propose a model to explanation the differences in replication observed between lambda phage and lambda plasmid DNA at low temperature. 相似文献