Degradation of thialysine- or selenalysine-containing abnormal proteins in CHO cells

CHO cells can incorporate thialysine and selenalysine in their proteins in substitution of lysine. Data are reported in the present paper showing that proteins containing either thialysine or selenalysine are unstable and quite rapidly degraded. The degradation rate is strictly related to the extent of protein lysine substitution. At similar extent of substitution, selenalysine-containing proteins are more unstable that thialysine-containing ones.     

Selenalysine utilization for growth and protein synthesis by a lysine requiring E. coli mutant

Summary Selenalysine can be utilized in substitution of lysine by a lysine requiring E. coli mutant. The presence of some lysine in the culture medium is necessary to allow selenalysine utilization for growth; in the presence of an excess of lysine, selenalysine is not utilized. When utilized, selenalysine gives rise to an increase of final growth. However, it shows some toxic effects as demonstrated by the decrease of both growth rate and cell viability. Selenalysine is incorporated into proteins in substitution of lysine. Up to a maximum of 50% of total protein lysine can be substituted. The decrease of cell viability is correlated with the extent of lysine substitution.This paper is dedicated to Professor A. E. Braunstein on his 80th birthday.     

Thialysine and selenalysine incorporation into CHO cell proteins and its effects on cell growth

CHO cells can incorporate into proteins both thialysine and selenalysine when both are present together in the culture medium. Thialysine and selenalysine inhibit cell growth and cell viability. The inhibitory effect of either analog is additive. The inhibition of cell viability is related to the extent of protein lysine substitution by thialysine or selenalysine; it is however irrelevant whether lysine is substituted by one or the other analog or by both.     

Degradation of thialysine- or selenalysine-containing abnormal proteins in E. coli

Thialysine and selenalysine can be utilized for protein synthesis by lysine-requiring E. coli cells even in the absence of lysine. Protein synthesis has been determined as labeled leucine incorporation into acid-insoluble material, as increase of cell proteins and as protein-lysine substitution by the analog. Either analog can be incorporated into proteins, in the absence of lysine, for a limited time interval after which cells stop to duplicate. Proteins synthesized during this period contain most of their lysine residues substituted by the analog. Moreover, it has been shown that the analog-containing proteins are unstable and rapidly degraded. Their instability would account for the inability of lysine-requiring E. coli cells to utilize the analog as growth factor.     

Utilization of lysine analogs by a lysine-requiring E. coli mutant

Thialysine and selenalysine cannot substitute lysine as a growth factor for a lysine-requiring E. coli mutant, but can nevertheless be utilized for protein synthesis in the presence of lysine. In order to have information about the effects of lysine on the utilization of the two analogs, the extent of the incorporation of the three aminoacids into newly synthesized proteins has been determined. The analog starts to be utilized by cells growing in a medium containing either analog and lysine when lysine concentration becomes very low. Of the two analogs, thialysine is more easily utilized. In fact thialysine can be utilized when the lysine/thialysine ratio in the medium is 1/25. Selenalysine starts to be utilized when the lysine/selenalysine ratio is 1/200.     

Thialysine and selenalysine utilization for protein synthesis by E. coli in comparison with lysine

Utilization of thialysine and selenalysine for protein synthesis by a lysine requiring E. coli mutant was studied. Incorporation into proteins of thialysine or selenalysine, added to culture medium together with lysine, becomes evident when the amount of available lysine in the medium is highly reduced, that is the mutant utilizes the isologs only after all the available natural aminoacid has been utilized. Compared to selenalysine, thialysine is better utilized; when both isologs are present in the medium at equal concentrations, up to 46% of protein lysine is substituted by thialysine and only 12% by selenalysine.     

Aspartokinase III repression and lysine analogs utilization for protein synthesis

The extents of thialysine and selenalysine incorporation into cell proteins were compared in E. coli KL16 and in a mutant able to grow equally well in the presence or in the absence of both lysine analogs. The mutant differs from the parental strain in the repression of aspartokinase III (AKIII), the first enzyme of the lysine biosynthetic pathway. No analog incorporation into proteins was observed in mutant cells grown in the presence of either analog, whereas a marked analog incorporation was observed in the parental strain, where up to 17% and 12% of protein lysine can be substituted by thialysine and selenalysine respectively. In the parental strain grown in media containing either analog at different concentration the extent of analog incorporation into proteins is related to the extent of AKIII repression.     

Repression of lysine transport in E. coli KL16

Data reported in this paper show that both lysine transport systems in E. coli KL16 can be repressed by lysine and its isologs, thialysine and selenalysine, whereas they are not repressed by ornithine. The repression is specific on lysine transport systems; it is evident with 0.01 mM lysine or isolog concentration and reaches a maximum with 0.1 mM concentration. By comparing the extent of repression by lysine and its isologs, lysine gives the highest and selenalysine the lowest degree of repression. The shift from the repressed to the depressed state is rather immediate once the amino acid is removed from the culture medium.     

Selenalysine and protein synthesis.

Selenalysine is a lysine analog having the gamma-methylene group substituted by a selenium atom. It has been demonstrated that selenalysine is activated and transferred to tRNAlys by either Escherichia coli or rat liver aminoacyl-tRNA synthetases, and inhibits lysine incorporation into polypeptides in protein-synthesizing systems from E. coli, rat liver or rabbit reticulocytes. All tests were performed in comparison with thialysine, a lysine analog having the gamma-methylene group substituted by a sulfur atom. In all the reactions studied, both thialysine and selenalysine act as competitive inhibitors of lysine. With respect to thialysine, selenalysine act as competitive inhibitors of lysine. With respect to thialysine, selenalysine shows a slightly lower activity as lysine inhibitor.     

Intracellular transport of thialysine and selenalysine in CHO cells

The intracellular transport of thialysine and selenalysine in CHO cells has been studied. Data have been obtained indicating that the two lysine analogs can be transported by both the cationic aminoacid transport system and by the L transport system. The affinity of the cationic aminoacid transport system is similar for the two lysine analogs but lower than that for lysine and the affinity of the L transport system for the two lysine analogs is lower than that for leucine.     

Thialysine- and selenalysine-resistance in a E. coli mutant

A thialysine-resistant mutant of E. coli strain KL16 also shows a lower sensitivity to selenalysine, the lysine analog containing selenium. No difference between the mutant and the parental strain has been shown regarding the affinities of the transport systems and the lysyl-tRNA synthetase for selenalysine, thialysine and lysine as well as the inhibitory effects of these three aminoacids on the activity of the lysine biosynthetic pathway. A marked difference between the two strains has been evidenced in the AK III repression: in the mutant the repression by selenalysine, thialysine and lysine is much lower than in the parental strain.     

Thialysine utilization for protein synthesis by CHO cells

Chinese Hamster Ovary (CHO) cells utilize thialysine when added to the culture medium. Thialysine utilization is prevented by increasing lysine concentration in the medium, thus indicating that thialysine is utilized in substitution for and in competition with lysine. Almost all thialysine disappeared from the medium is recovered in cell protein hydrolysates. Thialysine is used for protein synthesis in substitution for lysine, and up to 10% of lysine can be substituted.     

Thialysine utilization by E. coli and its effects on cell growth

Summary Thialysine and selenalysine, two lysine isologs having the -methylene group substituted by a sulfur or a selenium atom, respectively, inhibit E. coli lysine-sensitive aspartokinase. The inhibition is specific, reversible and non-competitive. Compared to lysine, the two isologs have a less marked inhibitory effect, but show a similar homotropic cooperativity with a Hill's coefficient of about 2. The inhibition by each isolog is additive to that by lysine. Both compounds protect the enzyme against thermal inactivation. Overall, the data reported indicate that thialysine and selenalysine bind to the same allosteric site of lysine, the physiological modulator of the enzyme.     

Biochemical characterization of a thialysine-resistant clone of CHO cells

The intracellular transport and the activation of lysine, thialysine and selenalysine have been investigated in a thialysine-resistant CHO cell mutant strain in comparison with the parental strain. The cationic amino acid transport system responsible for the transport of these 3 amino acids shows no differences between the 2 strains as regards its affinity for each of these amino acids. On the other hand the Vmax of the transport system in the mutant is about double that in the parental strain. The lysyl-tRNA synthetase, assayed both as ATP = PPi exchange reaction and lysyl-tRNA synthesis, shows a lower affinity for thialysine and selenalysine than for lysine in both strains; in the mutant, however, the difference is even greater. Thus the thialysine resistance of the mutant is mainly due to the properties of its lysyl-tRNA synthetase, which shows a greater difference of the affinities for lysine and thialysine with respect to the parental strain.     

Transport systems for lysine, thialysine and selenalysine in E. coli KL16

Two lysine transport systems have been identified in E. coli KL16. They differ in their affinity for lysine, one showing a KM of 0.36 microM and the other a KM of 4.7 microM. Different compounds with chemical similarities to lysine were tested for their capacity to interfere with lysine transport. Among these only thialysine and selenalysine competitively inhibit lysine transport. The inhibition is on both transport systems. Thialysine shows a KI of 4 microM for the low affinity system and a KI of 8 microM for the high affinity system. Selenalysine shows values of 6 microM and 12 microM respectively.     

Thialysine utilization by a lysine-requiring Escherichia coli mutant

Summary Thialysine cannot completely substitute lysine as growth factor for a lysine-requiring E. coli mutant. However it can be utilized for growth in the presence of limiting amounts of lysine, in substitution of, and in competition with this latter. The effects of thialysine on growth rate, protein synthesis rate and cell viability, and its incorporation into proteins were studied in function of lysine and thialysine concentration in the culture media. Up to 60% of protein lysine substitution by thialysine is observed, without appreciable effects on cell viability.     

Changes in function but not oligomeric size are associated with αB-crystallin lysine substitution

αB-Crystallin, ubiquitously expressed in many tissues including the ocular lens, is a small heat shock protein that can prevent protein aggregation. A number of post-translation modifications are reported to modify αB-crystallin function. Recent studies have identified αB-crystallin lysine residues are modified by acetylation and ubiquitination. Therefore, we sought to determine the effects of lysine to alanine substitution on αB-crystallin functions including chaperone activity and modulation of actin polymerization. Analysis of the ten substitution mutants as recombinant proteins indicated all the proteins were soluble and formed oligomeric complexes similar to wildtype protein. Lysozyme aggregation induced by chemical treatment indicated that K82, K90, K121, K166 and K174/K175 were required for efficient chaperone activity. Thermal induction of γ-crystallin aggregation could be prevented by all αB-crystallin substitution mutants. These αB-crystallin mutants also were able to mediate wildtype levels of actin polymerization. Further analysis of two clones with either enhanced or reduced chaperone activity on individual client substrates or actin polymerization indicated both retained broad chaperone activity and anti-apoptotic activity. Collectively, these studies show the requirements for lysine residues in αB-crystallin function.     

Thialysine utilization for protein synthesis by an exponentially growing E. coli culture

The extent of protein lysine substitution by thialysine in E. coli cells grown in media containing the analog depends on the time interval the cells are grown in the presence of analog and on the analog concentration in the medium. By calculating the percent of lysine substitution in newly synthesized proteins it was shown that this reaches, after one cell doubling in the presence of analog, a maximum which is 17% in the cells grown with 0.1 or 0.2 mM thialysine and 8% in cells grown with 0.05 mM thialysine. Proteins synthesized in the presence of analog in the concentration range 0.05-0.2 mM show similar stability to those synthesized in the absence of analog. The extent of analog incorporation into newly synthesized proteins, as regards both the time course and the dependence on analog concentration in the medium, is strictly related to the extent of the repression of AK III, the first enzyme of lysine biosynthetic pathway.     

Proteins containing reductively aminated disaccharides: Synthesis and chemical characterization

Synthetic glycoproteins can be prepared by reductive amination of protein and reducing disaccharide in the presence of sodium cyanoborohydride. The reaction proceeds readily in aqueous solutions over a broad pH range to give high degrees of substitution. The degree of substitution can be determined by amino acid analysis, as the secondary amine linkage formed by reductive amination in stable to acid-catalyzed protein hydrolysis conditions. In order to demonstrate that coupling occurs to lysine residues, synthetic α-N-1-(1-deoxyglucitol)-lysine and ?-N-1-(1-deoxyglucitol)-lysine were prepared and compared with bovine serum albumin conjugates of maltose, cellobiose, lactose, and melibiose by amino acid analysis after acid hydrolysis. These studies demonstrate that the expected secondary amine linkages are formed with the ?-amino groups of lysine.     

Lysine 144, a ubiquitin attachment site in HIV-1 Nef, is required for Nef-mediated CD4 down-regulation

Nef is a HIV-1 accessory protein critical for the replication of the virus and the development of AIDS. The major pathological activity of Nef is the down-regulation of CD4, the primary receptor of HIV-1 infection. The mechanism underlying Nef-mediated CD4 endocytosis and degradation remains incompletely understood. Since protein ubiquitination is the predominant sorting signal in receptor endocytosis, we investigated whether Nef is ubiquitinated. The in vivo ubiquitination assay showed that both HIV-1 and SIV Nef proteins expressed in Jurkat T cells and 293T cells were multiple ubiquitinated by ubiquitin-His. The lysine-free HIV-1 Nef mutant (Delta10K) generated by replacing all 10 lysines with arginines was not ubiquitinated and the major ubiquitin-His attachment sites in HIV-1 Nef were determined to be lysine 144 (di-ubiquitinated) and lysine 204 (mono-ubiquitinated). Lysine-free HIV-1 Nef was completely inactive in Nef-mediated CD4 down-regulation, so was the Nef mutant with a single arginine substitution at K144 but not at K204. A mutant HIV-1 provirion NL4-3 with a single arginine substitution in Nef at K144 was also inactive in Nef-mediated CD4 down-regulation. Lysine-free Nef mutant reintroduced with lysine 144 (DeltaK10 + K144) was shown active in CD4 down-regulation. These data suggest that ubiquitination of Nef, particularly diubiquitination of the lysine 144, is necessary for Nef-mediated CD4 down-regulation.