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1.
Hyperthermia increases levels of nuclear-associated proteins in a manner that correlates with cell killing. If the increase in nuclear-associated proteins represents a lethal lesion then treatments that protect against killing by heat should reduce and/or facilitate the recovery of levels of the proteins in heated cells. This hypothesis was tested using three heat protection treatments: cycloheximide, D2O, and thermotolerance. All three treatments reduced levels of the proteins measured immediately following hyperthermia at 43.0 or 45.5 degrees C, with the greatest reduction occurring at 43.0 degrees C. In addition to reducing the proteins, thermotolerance facilitated the recovery of the proteins to control levels following hyperthermia. Thus thermotolerance may protect cells by both reducing the initial heat damage and facilitating recovery from that damage. Cycloheximide and D2O did not facilitate recovery of nuclear-associated proteins, suggesting that their protection against cytotoxicity related to the proteins resulted solely from their reduction of increases in levels of the proteins. All three treatments have been shown to stabilize cellular proteins against thermal denaturation. The results of this study suggest that the increase in nuclear-associated proteins may result from thermally denatured proteins adhering to the nucleus and that it is the ability of cycloheximide, D2O, and thermotolerance to thermostabilize proteins that reduces the increase in levels of the proteins within heated cells.  相似文献   

2.
Combining single molecule atomic force microscopy (AFM) and protein engineering techniques, here we demonstrate that we can use recombination-based techniques to engineer novel elastomeric proteins by recombining protein fragments from structurally homologous parent proteins. Using I27 and I32 domains from the muscle protein titin as parent template proteins, we systematically shuffled the secondary structural elements of the two parent proteins and engineered 13 hybrid daughter proteins. Although I27 and I32 are highly homologous, and homology modeling predicted that the hybrid daughter proteins fold into structures that are similar to that of parent protein, we found that only eight of the 13 daughter proteins showed beta-sheet dominated structures that are similar to parent proteins, and the other five recombined proteins showed signatures of the formation of significant alpha-helical or random coil-like structure. Single molecule AFM revealed that six recombined daughter proteins are mechanically stable and exhibit mechanical properties that are different from the parent proteins. In contrast, another four of the hybrid proteins were found to be mechanically labile and unfold at forces that are lower than the approximately 20 pN, as we could not detect any unfolding force peaks. The last three hybrid proteins showed interesting duality in their mechanical unfolding behaviors. These results demonstrate the great potential of using recombination-based approaches to engineer novel elastomeric protein domains of diverse mechanical properties. Moreover, our results also revealed the challenges and complexity of developing a recombination-based approach into a laboratory-based directed evolution approach to engineer novel elastomeric proteins.  相似文献   

3.
In the plasma membrane of animal cells, many membrane-spanning proteins exhibit lower lateral mobilities than glycosylphosphatidylinositol (GPI)-linked proteins. To determine if the GPI linkage was a major determinant of the high lateral mobility of these proteins, we measured the lateral diffusion of chimeric membrane proteins composed of normally transmembrane proteins that were converted to GPI-linked proteins, or GPI-linked proteins that were converted to membrane-spanning proteins. These studies indicate that GPI linkage contributes only marginally (approximately twofold) to the higher mobility of several GPI-linked proteins. The major determinant of the high mobility of these proteins resides instead in the extracellular domain. We propose that lack of interaction of the extracellular domain of this protein class with other cell surface components allows diffusion that is constrained only by the diffusion of the membrane anchor. In contrast, cell surface interactions of the ectodomain of membrane-spanning proteins exemplified by the vesicular stomatitis virus G glycoprotein reduces their lateral diffusion coefficients by nearly 10-fold with respect to many GPI-linked proteins.  相似文献   

4.
Proteins in general consist not only of globular structural domains (SDs), but also of intrinsically disordered regions (IDRs), i.e. those that do not assume unique three-dimensional structures by themselves. Although IDRs are especially prevalent in eukaryotic proteins, the functions are mostly unknown. To elucidate the functions of IDRs, we first divided eukaryotic proteins into subcellular localizations, identified IDRs by the DICHOT system that accurately divides entire proteins into SDs and IDRs, and examined charge and hydropathy characteristics. On average, mitochondrial proteins have IDRs more positively charged than SDs. Comparison of mitochondrial proteins with orthologous prokaryotic proteins showed that mitochondrial proteins tend to have segments attached at both N and C termini, high fractions of which are IDRs. Segments added to the N-terminus of mitochondrial proteins contain not only signal sequences but also mature proteins and exhibit a positive charge gradient, with the magnitude increasing toward the N-terminus. This finding is consistent with the notion that positively charged residues are added to the N-terminus of proteobacterial proteins so that the extended proteins can be chromosomally encoded and efficiently transported to mitochondria after translation. By contrast, nuclear proteins generally have positively charged SDs and negatively charged IDRs. Among nuclear proteins, DNA-binding proteins have enhanced charge tendencies. We propose that SDs in nuclear proteins tend to be positively charged because of the need to bind to negatively charged nucleotides, while IDRs tend to be negatively charged to interact with other proteins or other regions of the same proteins to avoid premature proteasomal degradation.  相似文献   

5.
Moonlighting proteins comprise a subset of multifunctional proteins that perform two or more biochemical functions that are not due to gene fusions, multiple splice variants, proteolytic fragments, or promiscuous enzyme activities. The project described herein focuses on a sub-set of moonlighting proteins that have a canonical biochemical function inside the cell and perform a second biochemical function on the cell surface in at least one species. The goal of this project is to consider the biophysical features of these moonlighting proteins to determine whether they have shared characteristics or defining features that might suggest why these particular proteins were adopted for a second function on the cell surface, or if these proteins resemble typical intracellular proteins. The latter might suggest that many other normally intracellular proteins found on the cell surface might also be moonlighting in this fashion. We have identified 30 types of proteins that have different functions inside the cell and on the cell surface. Some of these proteins are found to moonlight on the surface of multiple species, sometimes with different extracellular functions in different species, so there are a total of 98 proteins in the study set. Although a variety of intracellular proteins (enzymes, chaperones, etc.) are observed to be re-used on the cell surface, for the most part, these proteins were found to have physical characteristics typical of intracellular proteins. Many other intracellular proteins have also been found on the surface of bacterial pathogens and other organisms in proteomics experiments. It is quite possible that many of those proteins also have a moonlighting function on the cell surface. The increasing number and variety of known moonlighting proteins suggest that there may be more moonlighting proteins than previously thought, and moonlighting might be a common feature of many more proteins.  相似文献   

6.
A number of eubacterial species contain methyl-accepting taxis proteins that are antigenically and thus structurally related to the well-characterized methyl-accepting chemotaxis proteins of Escherichia coli. Recent studies of the archaebacterium Halobacterium halobium have characterized methyl-accepting taxis proteins that in some ways resemble and in other ways differ from the analogous eubacterial proteins. We used immunoblotting with antisera raised to E. coli transducers to probe shared structural features of methyl-accepting proteins from archaebacteria and eubacteria and found substantial antigenic relationships. This implies that the genes for the contemporary methyl-accepting proteins are related through an ancestral gene that existed before the divergence of arachaebacteria and eubacteria. Analysis by immunoblot of mutants of H. halobium defective in taxis revealed that some strains were deficient in covalent modification of methyl-accepting proteins although the proteins themselves were present, while other strains appeared to be missing specific methyl-accepting proteins.  相似文献   

7.
Lysosomal membrane proteins and soluble lysosomal material were isolated from pulse-chase labelled human skin fibroblasts and examined for incorporation of radioactivity and affinity to immobilized mannose-6-phosphate-specific receptors. Incorporation of radioactivity into lysosomal membrane proteins was delayed by about 2 h on average when compared to that of soluble lysosomal proteins. The lack of binding indicates that a mannose-6-phosphate-independent mechanism is responsible for targeting of lysosomal membrane proteins to lysosomes. In contrast to soluble lysosomal proteins, the membrane proteins did not bind to mannose-6-phosphate specific receptors. The delayed appearance of membrane proteins in lysosomes as compared to that of soluble lysosomal proteins suggested that different pathways are utilized by the two classes of lysosomal proteins.  相似文献   

8.
Protein domain frequency and distribution among kingdoms was statistically analyzed using the SCOP structural database. It appeared that among chosen protein domains with the best resolution, eukaryotic proteins more often belong to α-helical and β-structural proteins, while proteins of bacterial origin belong to α/β structural class. Statistical analysis of folding rates of 73 proteins with known experimental data revealed that bacterial proteins with simple kinetics (23 proteins) exhibit a higher folding rate compared to eukaryotic proteins with simple folding kinetics (27 proteins). Analysis of protein domain amino acid composition showed that the frequency of amino acid residues in proteins of eukaryotic and bacterial origin is different for proteins with simple and complex folding kinetics.  相似文献   

9.
SR proteins are essential splicing factors required for constitutive splicing and function as key regulators of alternative RNA splicing. We have shown that SR proteins purified from late adenovirus-infected cells (SR-Ad) are functionally inactivated as splicing enhancer or splicing repressor proteins by a virus-induced partial de-phosphorylation. Here, we show that SR proteins purified from late vaccinia-virus-infected cells (SR-VV) are also hypo-phosphorylated and functionally inactivated as splicing regulatory proteins. We further show that incubating SR-Ad proteins under conditions that restore the phospho-epitopes to the SR proteins results in the restoration of their activity as splicing enhancer and splicing repressor proteins. Interestingly, re-phosphorylation of SR-VV proteins only partially restored the splicing enhancer or splicing repressor phenotype to the SR proteins. Collectively, our results suggest that viral control of SR protein activity may be a common strategy used by DNA viruses to take control of the host cell RNA splicing machinery.  相似文献   

10.
Repeat proteins have become increasingly important due to their capability to bind to almost any proteins and the potential as alternative therapy to monoclonal antibodies. In the past decade repeat proteins have been designed to mediate specific protein-protein interactions. The tetratricopeptide and ankyrin repeat proteins are two classes of helical repeat proteins that form different binding pockets to accommodate various partners. It is important to understand the factors that define folding and stability of repeat proteins in order to prioritize the most stable designed repeat proteins to further explore their potential binding affinities. Here we developed distance-dependant statistical potentials using two classes of alpha-helical repeat proteins, tetratricopeptide and ankyrin repeat proteins respectively, and evaluated their efficiency in predicting the stability of repeat proteins. We demonstrated that the repeat-specific statistical potentials based on these two classes of repeat proteins showed paramount accuracy compared with non-specific statistical potentials in: 1) discriminate correct vs. incorrect models 2) rank the stability of designed repeat proteins. In particular, the statistical scores correlate closely with the equilibrium unfolding free energies of repeat proteins and therefore would serve as a novel tool in quickly prioritizing the designed repeat proteins with high stability. StaRProtein web server was developed for predicting the stability of repeat proteins.  相似文献   

11.
Nonmotile mutants of the gliding bacterium Cytophaga johnsonae were examined to identify proteins that might be involved in gliding motility. Wild-type and mutant cell proteins were solubilized and fractionated by using Triton X-114, and the proteins that partitioned into the aqueous phase or the detergent phase were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for proteins that differed between wild-type and mutant cells. Seventeen proteins, ranging in size from 16 to 150 kilodaltons, were implicated by this technique as having some relationship to gliding and were designated Gld-1 through Gld-17. All Gld proteins behaved as integral membrane proteins, partitioning into the detergent phase. All 56 mutants examined exhibited changes in 1 or more of the Gld proteins, with the number of proteins altered in any mutant varying from 1 to 11. Several lines of evidence suggested that proteins Gld-12 through Gld-15 are glycoproteins. Analysis of banding patterns of detergent-fraction proteins of motile revertants supported the idea that the Gld proteins have a role in gliding motility.  相似文献   

12.
Surface proteins play important pathophysiological roles in health and disease, and accumulating proteomics-based studies suggest that several "non-membrane" proteins are sorted to the cell surface by unconventional mechanisms. Importantly, these proteins may comprise attractive therapeutic targets and novel disease markers for colon cancer. To perform a proteomics-based inventory of these so-called "anchorless" surface proteins, intact colon adenocarcinoma SW480 cells were labeled with membrane-impermeable biotin after which only soluble biotinylated proteins were isolated and identified by nanoLC-MS/MS. Computer-assisted analysis predicted that only 9 of the 97 identified surface-exposed proteins have predicted secretory signal peptides, whereas 2 other proteins have a putative transmembrane segment. Of the 9 proteins with putative signal peptides, 1 was predicted to be retained at the cell surface by a GPI-anchor, whereas 5 other proteins contained an ER-retention motif (KDEL) that should prevent them from being sorted to the cell surface. The remaining 86 soluble "surface" proteins lack known export signals and the possibility that these proteins are candidate substrates of non-classical transporters or exported by unconventional mechanisms is discussed. Alternatively, the large number of "intracellular" and ER-resident proteins may imply that biotinylation approaches are not only specific for surface proteins, but also biased against a certain subset of non-surface proteins. This underscores the importance of post-proteomic verification of proteomics-based inventories on surface-exposed proteins, which eventually should reveal to which extent non-classical export and retention mechanisms contribute to the sorting of "anchorless" proteins to the surface of colon tumor cells.  相似文献   

13.
For many years, heat shock or stress proteins have been regarded as intracellular molecules that have a range of housekeeping and cytoprotective functions, only being released into the extracellular environment in pathological situations such as necrotic cell death. However, evidence is now accumulating to indicate that, under certain circumstances, these proteins can be released from cells in the absence of cellular necrosis, and that extracellular heat shock proteins have a range of immunoregulatory activities. The capacity of heat shock proteins to induce pro-inflammatory responses, together with the phylogenetic similarity between prokaryotic and eukaryotic heat shock proteins, has led to the proposition that these proteins provide a link between infection and autoimmune disease. Indeed, both elevated levels of antibodies to heat shock proteins and an enhanced immune reactivity to heat shock proteins have been noted in a variety of pathogenic disease states. However, further evaluation of heat shock protein reactivity in autoimmune disease and after transplantation has shown that, rather than promoting disease, reactivity to self-heat shock proteins can downregulate the disease process. It might be that self-reactivity to heat shock proteins is a physiological response that regulates the development and progression of pro-inflammatory immunity to these ubiquitously expressed molecules. The evolving evidence that heat shock proteins are present in the extracellular environment, that reactivity to heat shock proteins does not necessarily reflect adverse, pro-inflammatory responses and that the promotion of reactivity to self-heat shock proteins can downregulate pathogenic processes all suggest a potential role for heat shock proteins as therapeutic agents, rather than as therapeutic targets.  相似文献   

14.
15.
Previous studies demonstrated that palmitate and myristate are covalently linked to distinct sets of cellular proteins and that the linkages through which these fatty acids are attached to the polypeptide chains are different (Olson, E. N., Towler, D. A., and Glaser, L. (1985) J. Biol. Chem. 260, 3784-3790). In the present study, the kinetics and subcellular sites of acylation of proteins with palmitate and myristate were examined in the BC3H1 muscle cell line. Acylation with myristate was an extremely early modification that appeared to take place cotranslationally or shortly thereafter for a variety of soluble and membrane-bound proteins. In contrast, acylation of proteins with palmitate was a post-translational event that occurred exclusively on membrane proteins. To begin to understand the intracellular pathways that acyl proteins follow during their maturation, the degree of glycosylation, and the nature of the interaction of these proteins with membranes were examined. The majority of acyl proteins were tightly associated with membranes and could not be removed by conditions that release peripheral proteins from membranes. However, only a minor fraction of acylated proteins were N-glycosylated. These data suggest that the acyltransferases that attach palmitate and myristate to proteins are present in different subcellular locations and demonstrate that these fatty acids are attached to newly synthesized acyl proteins at different times during their maturation. The lack of carbohydrate on the majority of integral membrane acyl proteins suggests that these proteins may follow intracellular pathways that are different from those followed by cell surface glycoproteins.  相似文献   

16.
I selected 82 proteins that were related to amino acid biosynthesis in the genome of Escherichia coli. I then searched the extensive sequence homology for each of the selected proteins from among the proteins of E.coli. The result showed that 30 proteins of the selected proteins had extensive sequence homology within the selected proteins, and 21 proteins had extensive sequence homology to proteins outside the selected proteins. In addition, the enzymes with broad substrate specificity play an important role in the amino acid biosynthesis. I demonstrate here that some substrate-specific enzymes evolved from an ancestor enzyme with broad substrate specificity. CONTACT: hnishida@iam.u-tokyo.ac.jp  相似文献   

17.
Cation-pi interactions play an important role to the stability of protein structures. In this work, we analyze the influence of cation-pi interactions in three-dimensional structures of membrane proteins. We found that transmembrane strand (TMS) proteins have more number of cation-pi interactions than transmembrane helical (TMH) proteins. In TMH proteins, both the positively charged residues Lys and Arg equally experience favorable cation-pi interactions whereas in TMS proteins, Arg is more likely than Lys to be in such interactions. There is no relationship between number of cation-pi interactions and number of residues in TMH proteins whereas a good correlation was observed in TMS proteins. The average cation-pi interaction energy for TMH proteins is -16 kcal/mol and that for TMS proteins is -27 kcal/mol. The pair-wise cation-pi interaction energy between aromatic and positively charged residues showed that Lys-Trp energy is stronger in TMS proteins than TMH proteins; Arg-Phe, Arg-Tyr and Lys-Phe have higher energy in TMH proteins than TMS proteins. The decomposition of energies into electrostatic and van der Waals revealed that the contribution from electrostatic energy is twice as that from van der Waals energy in both TMH and TMS proteins. The results obtained in the present study would be helpful to understand the contribution of cation-pi interactions to the stability of membrane proteins.  相似文献   

18.
Gene activation and DNA binding by Drosophila Ubx and abd-A proteins   总被引:18,自引:0,他引:18  
M L Samson  L Jackson-Grusby  R Brent 《Cell》1989,57(6):1045-1052
The Ubx and abd-A gene products are required for proper development of thoracic and abdominal structures in Drosophila. We expressed LexA-Ubx and LexA-abdA fusion proteins in yeast. These proteins activated expression of target genes that carried either upstream LexA operators or upstream Ubx binding sites. Both proteins contain homeodomains. Experiments with mutant fusion proteins show that the homeodomain is not required for the proteins to form dimers or enter the nucleus, and that, when DNA binding is provided by the LexA moiety, the homeodomain is not required for gene activation. Our results suggest that the homeodomain is necessary for these proteins to bind Ubx sites, but that the homeodomain does not contact DNA exactly like bacterial helix-turn-helix proteins. Finally, our data suggest that gene activation by these proteins is a simple consequence of their binding to DNA, while negative gene regulation requires that these proteins act together with other Drosophila gene products.  相似文献   

19.
Recent developments in research on the stability of proteins - specifically, comparisons of the ion pairs of homologous structures - show that ion pairs potentially contribute to the thermostability of proteins. This study proposes a probabilistic Bayesian statistical method to efficiently predict the thermostability of proteins based on the properties of ion pairs. The experimental results suggest that the numbers, types and bond lengths of ion pairs can be used to predict with high accuracy (up to 80%) the thermostability of functionally similar proteins. The predictions have high precision (99%), especially for hyperthermophilic proteins. Results for proteins with differing functions also indicate that the number of ion pairs is related to the thermostability of proteins, and that predictions of thermostability can also be made for proteins with different functions.  相似文献   

20.
During cold acclimation, winter rye (Secale cereale L.) plants secrete antifreeze proteins that are similar to pathogenesis-related (PR) proteins. In this experiment, the secretion of PR proteins was induced at warm temperatures by infection with pink snow mold (Microdochium nivale), a pathogen of overwintering cereals. A comparison of cold-induced and pathogen-induced proteins showed that PR proteins accumulated in the leaf apoplast to a greater level in response to cold. The PR proteins induced by cold and by snow mold were similar when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by immunoblotting. Both groups of PR proteins contained glucanase-like, chitinase-like, and thaumatin-like proteins, and both groups exhibited similar levels of glucanase and chitinase activities. However, only the PR proteins induced by cold exhibited antifreeze activity. Our findings suggest that the cold-induced PR proteins may be isoforms that function as antifreeze proteins to modify the growth of ice during freezing while also providing resistance to the growth of low-temperature pathogens in advance of infection. Both functions of the cold-induced PR proteins may improve the survival of overwintering cereals.  相似文献   

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