Age-Induced Protein Modifications and Increased Proteolysis in Potato Seed-Tubers

Long-term aging of potato (Solanum tuberosum) seed-tubers resulted in a loss of patatin (40 kD) and a cysteine-proteinase inhibitor, potato multicystatin (PMC), as well as an increase in the activities of 84-, 95-, and 125-kD proteinases. Highly active, additional proteinases (75, 90, and 100 kD) appeared in the oldest tubers. Over 90% of the total proteolytic activity in aged tubers was sensitive to trans-epoxysuccinyl-l-leucylamido (4-guanidino) butane or leupeptin, whereas pepstatin was the most effective inhibitor of proteinases in young tubers. Proteinases in aged tubers were also inhibited by crude extracts or purified PMC from young tubers, suggesting that the loss of PMC was responsible for the age-induced increase in proteinase activity. Nonenzymatic oxidation, glycation, and deamidation of proteins were enhanced by aging. Aged tubers developed “daughter” tubers that contained 3-fold more protein than “mother” tubers, with a polypeptide profile consistent with that of young tubers. Although PMC and patatin were absent from the older mother tubers, both proteins were expressed in the daughter tubers, indicating that aging did not compromise the efficacy of genes encoding PMC and patatin. Unlike the mother tubers, proteinase activity in daughter tubers was undetectable. Our results indicate that tuber aging nonenzymatically modifies proteins, which enhances their susceptibility to breakdown; we also identify a role for PMC in regulating protein turnover in potato tubers.Potato (Solanum tuberosum) seed-tubers are a model system for studying the process of aging in plants. The tubers can be stored (at 4°C and 95% RH) for to 3 years without a loss of viability. However, storage (aging) beyond about 8 months effects a progressive decline in apical dominance, rooting ability, and sprout vigor (Kumar and Knowles, 1993a). In addition to changes in growth potential, aging is accompanied by increased respiration of tubers (Kumar and Knowles, 1996a), oxidative stress (Kumar and Knowles, 1996b), lipid peroxidation (Kumar and Knowles, 1993b), and decreased protein content (Kumar and Knowles, 1993c). Although protein loss is partly due to reduced synthesis (Kumar and Knowles, 1993c), the contribution of proteolysis and the mechanisms by which proteins become damaged and subsequently targeted for degradation with advancing age are unknown. Processes that may lead to protein degradation during aging include (a) increased accessibility of proteins to proteinases resulting from decompartmentation, (b) molecular modifications to polypeptides that enhance proteolysis, and (c) increased activity of proteinases (Dalling, 1987).Oxidation, glycation, and isomerization/racemization of amino acid residues of proteins have been identified as nonenzymatic mechanisms that can adversely affect structure and function (Fig. ​(Fig.1),1), rendering proteins more susceptible to proteolysis during aging (Dalling, 1987; Stadtman, 1992; Luthra and Balasubramanian, 1993; Eckardt and Pell, 1995). Oxidative stress contributes to the formation of carbonyl derivatives on amino acid residues of proteins (Dalling, 1987; Oliver et al., 1987; Levine et al., 1990). For example, carbonyl content and susceptibility of Rubisco to proteolysis increased during oxidative stress (Ferriera and Shaw, 1989; Penarrubia and Moreno, 1990; Garcia-Ferris and Moreno, 1993; Eckardt and Pell, 1995). Similarly, oxidative stress caused by the inhibition of catalase by aminotriazole in maize seedlings resulted in a 2-fold increase in protein carbonyl content (Prasad, 1997). The increased oxidative stress accompanying aging of potato tubers may provide an ideal environment for oxidation of proteins. Figure 1Schematic diagram showing several nonenzymatic mechanisms that could affect protein structure and function in aging potato tubers. Protein modifications that may accompany aging include oxidation (increased carbonyl groups), glycation (reaction of ...Amino groups of proteins can react with aldehyde or keto groups of reducing sugars through a Schiff-base reaction, yielding brown fluorescent pigments known as advanced glycation end products (Luthra and Balasubramaniyan, 1993). Proteins thus modified tend to form cross-links (Fig. ​(Fig.1)1) that can destroy protein function (Wettlaufer and Leopold, 1991). A number of age-related diseases in humans are attributed to protein glycation. For example, in diabetics, elevated blood Glc is associated with cataracts (Monnier et al., 1979), accelerated aging, and vascular narrowing (Brownlee et al., 1986; Cerami et al., 1987). In light of the substantial increase in reducing sugar concentration of tubers during aging (Kumar and Knowles, 1993b), it was of interest to determine the extent of age-induced protein glycation.Proteins are also susceptible to nonenzymatic modification by deamidation-mediated conversion of l-asparaginyl to l-isoaspartyl residues (Fig. ​(Fig.1).1). Although proteins containing isomerized residues can be targeted for degradation, they are also substrates for PCMT (type II), which can restore protein function. Repair to such damaged proteins involves methylation, using AdoMet as a methyl donor. PCMT is a cytosolic “housekeeping” enzyme with specificity for the recognition and repair of altered aspartyl residues (Galletti et al., 1995), and has been detected in 45 plant species belonging to 23 families (Mudgett et al., 1997). Changes in PCMT activity with advancing tuber age were thus characterized as an indicator of deamidation-mediated damage to proteins.In addition to reduced protein synthesis and enhanced susceptibility of proteins to proteolysis, advancing tuber age may contribute to loss in the ability to synthesize proteinase inhibitors and thus to protein catabolism. Potato tubers contain a proteinase inhibitor, PMC (Rodis and Hoff, 1984; Walsh and Strikland, 1993). With its multiple inhibitory domains, PMC (85 kD) has the capacity for simultaneous inhibition of several Cys-proteinase molecules (Walsh and Strickland, 1993). The effect of aging on PMC and proteinase levels is unknown. Using potato as a model system, we examined potential mechanisms for age-induced protein loss and the extent to which proteins become nonenzymatically modified during aging.     

Rubisco small and large subunit N-methyltransferases. Bi- and mono-functional methyltransferases that methylate the small and large subunits of Rubisco

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)is methylated at the alpha-amino group of the N-terminal methionine of the processed form of the small subunit (SS), and at the epsilon-amino group of lysine-14 of the large subunit (LS) in some species. The Rubisco LS methyltransferase (LSMT) gene has been cloned and expressed from pea and specifically methylates lysine-14 of the LS of Rubisco. We determine here that both pea and tobacco Rubisco LSMT also exhibit (alpha)N-methyltransferase activity toward the SS of Rubisco, suggesting that a single gene product can produce a bifunctional protein methyltransferase capable of catalyzing both (alpha)N-methylation of the SS and (epsilon)N-methylation of the LS. A homologue of the Rubisco LSMT gene (rbcMT-S) has also been identified in spinach that is closely related to Rubisco LSMT sequences from pea and tobacco. Two mRNAs are produced from rbcMT-S, and both long and short forms of the spinach cDNAs were expressed in Escherichia coli cells and shown to catalyze methylation of the alpha-amino group of the N-terminal methionine of the SS of Rubisco. Thus, the absence of lysine-14 methylation in species like spinach is apparently a consequence of a monofunctional protein methyltransferase incapable of methylating Lys-14, with activity limited to methylation of the SS.     

Kinetic manifestation of processivity during multiple methylations catalyzed by SET domain protein methyltransferases

Processive versus distributive methyl group transfer was assessed for pea Rubisco large subunit methyltransferase, a SET domain protein lysine methyltransferase catalyzing the formation of trimethyllysine-14 in the large subunit of Rubisco. Catalytically competent complexes between an immobilized form of des(methyl) Rubisco and Rubisco large subunit methyltransferase were used to demonstrate enzyme release that was co-incident with and dependent on formation of trimethyllysine. Catalytic rate constants determined for formation of trimethyllysine were considerably lower ( approximately 10-fold) than rate constants determined for total radiolabel incorporation from [3H-methyl]-S-adenosylmethionine. Double-reciprocal velocity plots under catalytic conditions favoring monomethyllysine indicated a random or ordered reaction mechanism, while conditions favoring trimethyllysine suggested a hybrid ping-pong mechanism. These results were compared with double-reciprocal velocity plots and product analyses obtained for HsSET7/9 (a monomethyltransferase) and SpCLR4 (a dimethyltransferase) and suggest a predictive ability of double-reciprocal velocity plots for single versus multiple methyl group transfers by SET domain protein lysine methyltransferases. A model is proposed for SET domain protein lysine methyltransferases in which initial binding of polypeptide substrate and S-adenosylmethionine is random, with polypeptide binding followed by deprotonation of the epsilon-amine of the target lysyl residue and subsequent methylation. Following methyl group transfer, S-adenosylhomocysteine and monomethylated polypeptide dissociate from monomethyltransferases, but di- and trimethyltransferases begin a successive and catalytically obligatory deprotonation of enzyme-bound methylated lysyl intermediates, which along with binding and release of S-adenosylmethionine and S-adenosylhomocysteine is manifested as a hybrid ping-pong-like reaction mechanism.     

Posttranslational modifications in the amino- terminal region of the large subunit of ribulose- 1,5-bisphosphate carboxylase/oxygenase from several plant species

A combination of limited tryptic proteolysis, reverse phasehigh performance liquid chromatography, Edman degradative sequencing, amino acid analysis, and fast-atom bombardment mass-spectrometry was used to remove and identify the first 14 to 18 N-terminal amino acid residues of the large subunit of higher plant-type ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Chlamydomonas reinhardtii, Marchantia polymorpha, pea (Pisum sativum), tomato (Lycopersicon esculentum), potato (Solanum tuberosum), pepper (Capsicum annuum), soybean (Glycine max), petunia (Petunia x hybrida), cowpea (Vigna sinensis), and cucumber (Cucumis sativus) plants. The N-terminal tryptic peptide from acetylated Pro-3 to Lys-8 of the large subunit of Rubisco was identical in all species, but the amino acid sequence of the penultimate N-terminal tryptic peptide varied. Eight of the 10 species examined contained a trimethyllysyl residue at position 14 in the large subunit of Rubisco, whereas Chlamydomonas and Marchantia contained an unmodified lysyl residue at this position.     

Electrophysiological and biochemical responses of mouse vomeronasal receptor cells to urine-derived compounds: possible mechanism of action

Receptor cells of the vomeronasal organ (VNO) are thought to detect pheromone-like molecules important for reproductive physiology. Several compounds derived from male mouse urine have been demonstrated to affect endocrine events in female mice. In the present study, the ability of these compounds to affect VNO activity was tested. In dissociated VNO cells held under voltage clamp conditions, application of dehydro-exo-brevicomin (DHB) evoked an outward current at negative holding potentials and an inward current at positive holding potentials. Under current clamp, DHB reduced action potential firing. Since DHB application caused a decrease in membrane conductance, this compound appeared to act by reducing inward current through closing an ion channel. Biochemical experiments tested the effects of DHB and 2- (sec-butyl)-4,5-dihydrothiazole (SBT) on cAMP levels in the VNO. A mixture of DHB and SBT decreased cAMP levels in VNO sensory tissue and had no effect on VNO non-sensory tissue. The results suggest that pheromones have an inhibitory influence on action potential generation and on cAMP levels in receptor cells of the VNO.      

Antibodies designed as effective cancer vaccines

Antigen/antibody complexes can efficiently target antigen presenting cells to allow stimulation of the cellular immune response. Due to the difficulty of manufacture and their inherent instability complexes have proved inefficient cancer vaccines. However, anti-idiotypic antibodies mimicking antigens have been shown to stimulate both antibody and T cell responses. The latter are due to T cell mimotopes expressed within the complementarity-determining regions (CDRs) of antibodies that are efficiently presented to dendritic cells in vivo. Based on this observation we have designed a DNA vaccine platform called ImmunoBody™, where cytotoxic T lymphocyte (CTL) and helper T cell epitopes replace CDR regions within the framework of a human IgG1 antibody. The ImmunoBody™ expression system has a number of design features which allow for rapid production of a wide range of vaccines. The CDR regions of the heavy and light chain have been engineered to contain unique restriction endonuclease sites, which can be easily opened, and oligonucleotides encoding the T cell epitopes inserted. The variable and constant regions of the ImmunoBody™ are also flanked by restriction sites, which permit easy exchange of other IgG subtypes. Here we show a range of T cell epitopes can be inserted into the ImmunoBody™ vector and upon immunization these T cell epitopes are efficiently processed and presented to stimulate high frequency helper and CTL responses capable of anti-tumor activity.Key words: DNA vaccines, cancer vaccines, melanoma, CTL, helper T cells     

Trends in lizard translocations in New Zealand between 1988 and 2013

There is growing concern about mitigation-driven translocations that move animals from anthropogenic threats at donor sites because of their failure rate and lack of application of scientific principles and best practice. We reviewed all known lizard translocations in New Zealand between 1988 and 2013 and identified 85 translocations of 30 lizard taxa to 46 release sites. Most translocations (62%) were motivated by conservation goals for the species or the release site, and one-third were mitigation-driven translocations, typically motivated by habitat loss due to development. Mitigation-driven translocations began in 2003, and since that time have equalled the number of conservation-motivated translocations. Conservation-motivated translocations usually released lizards on islands without mammalian predators, whereas mitigation-driven translocations usually relocated lizards to mainland sites with introduced predators. Long-term monitoring has been sparse and often rudimentary. Eight lizard translocations have recorded population growth, including one mitigation-driven translocation that was into a fenced reserve. Research on commonly used management techniques to mitigate human-related impacts is recommended to establish whether these techniques benefit lizards in the long term.     

The pKO2 linear plasmid prophage of Klebsiella oxytoca

Temperate bacteriophages with plasmid prophages are uncommon in nature, and of these only phages N15 and PY54 are known to have a linear plasmid prophage with closed hairpin telomeres. We report here the complete nucleotide sequence of the 51,601-bp Klebsiella oxytoca linear plasmid pKO2, and we demonstrate experimentally that it is also a prophage. We call this bacteriophage phiKO2. An analysis of the 64 predicted phiKO2 genes indicate that it is a fairly close relative of phage N15; they share a mosaic relationship that is typical of different members of double-stranded DNA tailed-phage groups. Although the head, tail shaft, and lysis genes are not recognizably homologous between these phages, other genes such as the plasmid partitioning, replicase, prophage repressor, and protelomerase genes (and their putative targets) are so similar that we predict that they must have nearly identical DNA binding specificities. The phiKO2 virion is unusual in that its phage lambda-like tails have an exceptionally long (3,433 amino acids) central tip tail fiber protein. The phiKO2 genome also carries putative homologues of bacterial dinI and umuD genes, both of which are involved in the host SOS response. We show that these divergently transcribed genes are regulated by LexA protein binding to a single target site that overlaps both promoters.     

The generalized transducing Salmonella bacteriophage ES18: complete genome sequence and DNA packaging strategy

The generalized transducing double-stranded DNA bacteriophage ES18 has an icosahedral head and a long noncontractile tail, and it infects both rough and smooth Salmonella enterica strains. We report here the complete 46,900-bp genome nucleotide sequence and provide an analysis of the sequence. Its 79 genes and their organization clearly show that ES18 is a member of the lambda-like (lambdoid) phage group; however, it contains a novel set of genes that program assembly of the virion head. Most of its integration-excision, immunity, Nin region, and lysis genes are nearly identical to those of the short-tailed Salmonella phage P22, while other early genes are nearly identical to Escherichia coli phages lambda and HK97, S. enterica phage ST64T, or a Shigella flexneri prophage. Some of the ES18 late genes are novel, while others are most closely related to phages HK97, lambda, or N15. Thus, the ES18 genome is mosaically related to other lambdoid phages, as is typical for all group members. Analysis of virion DNA showed that it is circularly permuted and about 10% terminally redundant and that initiation of DNA packaging series occurs across an approximately 1-kbp region rather than at a precise location on the genome. This supports a model in which ES18 terminase can move substantial distances along the DNA between recognition and cleavage of DNA destined to be packaged. Bioinformatic analysis of large terminase subunits shows that the different functional classes of phage-encoded terminases can usually be predicted from their amino acid sequence.