Modulation Of Protein Levels Research Articles

Prognostic Significance of Both the Cytoplasmic and Nuclear Overexpression of Yin-Yang 1 (YY1) among Patients with Multiple Myeloma (MM)

Objective and Rationale Yin-Yang 1 (YY1) is a multi-functional DNA-binding protein, which can activate, repress or initiate transcription depending on the context in which it binds. In addition, YY1 can modulate protein levels or activity through protein-protein interaction. YY1 has been identified as a potential repressor factor for several genes. We have reported that YY1 can act as a transcription repressor for both Fas and TRAIL DR5. In addition to YY1-mediated regulation of tumor cell resistance to cytotoxic immunotherapy, it also has been shown to regulate resistance to chemotherapy [Baritaki et al., J Immunol 80:6199,2008]. Overexpression of YY1 has been shown to be of prognostic significance in prostate cancer [Seligson et al., Int J Oncol 27:131,2005].Hypothesis In this study, we have examined the expression of YY1 in MM (cell lines and patients' bone marrow [BM]) by hypothesizing that the resistance of MM cells to various cytotoxic agents may be, in part, regulated by overexpression of YY1 and that YY1 may also be of prognostic significance.Designs and Methods MM cell lines and fresh BM samples from MM patients (n=21) were examined for YY1 expression, cytoplasmic and nuclear, by immunohistochemistry and by Western. The specificity of the anti-YY1 antibody was demonstrated by the competitive inhibition with a peptide used for immunization. Control isotype IgG did not show any staining on the cells.Results First, we found that various MM cell lines (RMPI 8226, IM-9, U266) overexpress YY1 both in the cytoplasm and the nucleus. Next, we examined the expression of YY1 in BM derived from MM patients by immunohistochemistry. BM from MM patients showed overexpression of YY1 as compared to normal bone marrow samples. Furthermore, analysis of both the intensity and frequency of cells expressing YY1 in both the cytoplasm and nucleus was shown to be significantly higher among patients with progressive disease as compared to patients with stable or responsive disease.Conclusions and Implications These findings show that the expression of YY1 among patients with MM may correlate with progression and also suggest the prognostic significance of YY1 in MM patients. Inhibition of YY1 by various agents (example: low-dose chemotherapy, proteasome inhibitors, NO donors, NF-kB inhibitors, etc.) all result in the reversal of resistance to various cytotoxic agents (eg CDDP, TRAIL) therefore, the findings also imply that agents that can inhibit YY1 expression in MM patients may be of therapeutic potential when used in combination with conventional therapeutics.

Mice lacking the kf-1 gene exhibit increased anxiety- but not despair-like behavior

KF-1 was originally identified as a protein encoded by human gene with increased expression in the cerebral cortex of a patient with Alzheimer's disease. In mouse brain, kf-1 mRNA is detected predominantly in the hippocampus and cerebellum, and kf-1 gene expression is elevated also in the frontal cortex of rats after chronic antidepressant treatments. KF-1 mediates E2-dependent ubiquitination and may modulate cellular protein levels as an E3 ubiquitin ligase, though its target proteins are not yet identified. To elucidate the role of kf-1 in the central nervous system, we generated kf-1 knockout mice by gene targeting, using Cre-lox recombination. The resulting kf-1−/− mice were normal and healthy in appearance. Behavioral analyses revealed that kf-1−/− mice showed significantly increased anxiety-like behavior compared with kf-1+/+ littermates in the light/dark transition and elevated plus maze tests; however, no significant differences were observed in exploratory locomotion using the open field test or in behavioral despair using the forced swim and tail suspension tests. These observations suggest that KF-1 suppresses selectively anxiety under physiological conditions probably through modulating protein levels of its unknown target(s). Interestingly, kf-1−/− mice exhibited significantly increased prepulse inhibition, which is usually reduced in human schizophrenic patients. Thus, the kf-1−/− mice provide a novel animal model for elucidating molecular mechanisms of psychiatric diseases such as anxiety/depression, and may be useful for screening novel anxiolytic/antidepressant compounds.

Characterization of a type A response regulator in the common bean (Phaseolus vulgaris) in response to phosphate starvation

Type A response regulators are a family of genes in Arabidopsis thaliana involved primarily in cytokinin signal transduction. A member of this family was isolated from a cDNA library constructed from bean plants (Phaseolus vulgaris) grown under conditions of phosphate starvation. The complete cDNA sequence showed the presence of the DDK domain, which is the hallmark of the response regulator family. Expression of the P. vulgaris response regulator 1 (PvRR1) showed clear regulation based on phosphate availability because transcript levels increased during phosphate starvation and returned to basal levels after resupplementation with phosphorus. Nitrogen and potassium starvation also upregulated PvRR1, indicating that cross talk with other nutrient signaling pathways might occur. Addition of cytokinins to plants growing under phosphate-sufficient conditions stimulated PvRR1 transcript levels both in detached leaves and in roots. However, cytokinins strongly inhibited PvRR1 expression in phosphate-starved plants after 24 h of incubation. At the protein level, subcellular localization of PvRR1 indicated that it is a nuclear protein and that phosphate starvation modified protein levels but not the localization.

Early Exposure to General Anesthesia Causes Significant Neuronal Deletion in the Developing Rat Brain

Frequent exposure of children to general anesthesia is common practice in modern medicine. Although previously unrecognized, recent in vitro and in vivo animal studies suggest that exposure to clinically relevant general anesthetics at the peak of brain development could be detrimental to immature mammalian neurons, as demonstrated by massive and widespread apoptotic neurodegeneration. The survival of the developing neurons presumably depends on proper and timely formation of synapses, for which synaptic proteins (e.g., synaptophysin, synaptobrevin, amphiphysin, synaptosomal-associated protein 25 [SNAP-25], and Ca(2+)/calmodulin-dependent protein kinase II [CaM kinase II]) are crucially important. Overinhibition of developing neurons impairs synaptic protein function and activity-induced synaptic plasticity, which could in turn result in permanent neuronal loss. To examine the effects of general anesthesia, the pharmacological agents known to cause extensive neuronal inhibition, on synaptic proteins, and neuronal survival at the peak of synaptogenesis, we exposed 7-day-old rat pups to general anesthesia (midazolam, 9 mg/kg of body weight, subcutaneously, followed by 6 h of nitrous oxide 75 vol% and isoflurane 0.75 vol%). We found that this general anesthesia causes permanent neuronal deletion in the most vulnerable brain regions-the cerebral cortex and the thalamus-while transiently modulating protein levels of synaptophysin, synaptobrevin, amphiphysin, SNAP-25, and CaM kinase II.

The sweet nature of cardioprotection

glucose, glutamine, and glucosamine are metabolites that can alter cardioprotection ([1][1], [3][2], [8][3], [9][4], [13][5]–[15][6]). Numerous studies suggest that one molecular mechanism by which these metabolites protect cells is though their conversion to uridine diphosphate- N -

Protective Effect of TEMPOL Derivatives against Light-Induced Retinal Damage in Rats

OT-551 (1-hydroxy-4-cyclopropanecarbonyloxy-2,2,6,6-tetramethylpiperidine hydrochloride), a TEMPOL-H (OT-674) derivative, is a new catalytic antioxidant. In the present study, the efficacy of OT-551 and OT-674 in retinal neuroprotection was tested in a model of light-induced photoreceptor degeneration. Albino rats were intraperitoneally injected with OT-551, OT-674, or water, approximately 30 minutes before a 6-hour exposure to 2700-lux white fluorescent light. Retinal protection was evaluated histologically by measuring the thickness of the outer nuclear layer (ONL) and functionally by electroretinogram (ERG) analysis, 5 to 7 days after exposure to light. Levels of protein modification by 4-hydroxynonenal (4-HNE) and 4-hydroxyhexenal (4-HHE), which are end products of the nonenzymatic oxidation of n-6 and n-3 polyunsaturated fatty acids, respectively, were measured by Western dot blot analysis immediately after exposure to light. After exposure to light, water-treated animals had a 77% loss of ERG b-wave amplitudes and a 26% and 56% loss of mean ONL thickness in the inferior and superior hemispheres, respectively. Compared with water-treated rats, ERG b-wave amplitudes in light-exposed eyes were significantly higher in 25 (P < 0.05)-, 50 (P < 0.05)-, and 100 (P < 0.001)-mg/kg OT-551-treated rats. Mean ONL thickness in the superior hemisphere was significantly higher in 25 (P < 0.01)-, 50 (P < 0.01)-, and 100 (P < 0.001)-mg/kg OT-551-treated, light-exposed eyes and in 100 mg/kg (P < 0.05) OT-674-treated eyes. No decrease of ONL thickness was observed in the light-protected covered fellow eyes in any animal. Increased levels of 4-HNE- and 4-HHE-protein modifications after exposure to light in water-treated eyes were completely counteracted by 100 mg/kg OT-551. Systemic administration of OT-551 and OT-674 provides both functional and morphologic photoreceptor cell protection against acute light-induced damage, most likely by inhibiting lipid peroxidation. The protection by OT-551 was greater than OT-674.

Maleic anhydride derivatives of a protein isolate: preparation and functional evaluation

African locust bean protein isolate (NPI) was acylated with maleic anhydride at various concentrations. Addition of maleic anhydride at the levels of 0.1, 0.5 and 1.0 g/g of protein produced 36.4% (MP1), 43.7% (MP2) and 73.6% (MP3) of maleylated protein isolate, respectively. Water absorption capacity increased after maleylation but oil absorption capacity reduced progressively with increase in level of modification. Results also revealed that gelation capacities reduced after protein modifications. Emulsifying activity (EA) and emulsion stability (ES) increased following protein modification. In addition, corresponding increases in emulsifying properties were observed with increase in degree of modification of the proteins. Maleylation improved the foaming capacity of the native protein. Foaming capacity increased with increases in protein concentration for both native and maleylated protein derivatives while progressive decline in foam stability occurred with increases in level of protein modification.

Poly(ADP-Ribose) Polymerase Inhibitors Ameliorate Nephropathy of Type 2 Diabetic Leprdb/dbMice

The activation of the poly(ADP-ribose) polymerase (PARP) plays an important role in the pathophysiology of various diseases associated with oxidative stress. We found increased amounts of poly(ADP) ribosylated proteins in diabetic kidneys of Lepr(db/db) (BKsJ) mice, suggesting increased PARP activity. Therefore, we examined the effects of two structurally unrelated PARP inhibitors (INO-1001 and PJ-34) on the development of diabetic nephropathy of Lepr(db/db) (BKsJ) mice, an experimental model of type 2 diabetes. INO-1001 and PJ-34 were administered in the drinking water to Lepr(db/db) mice. Both INO-1001 and PJ-34 treatment ameliorated diabetes-induced albumin excretion and mesangial expansion, which are hallmarks of diabetic nephropathy. PARP inhibitors decreased diabetes-induced podocyte depletion in vivo and blocked hyperglycemia-induced podocyte apoptosis in vitro. High glucose treatment of podocytes in vitro led to an early increase of poly(ADP) ribosylated modified protein levels. Reactive oxygen species (ROS) generation appears to be a downstream target of hyperglycemia-induced PARP activation, as PARP inhibitors blocked the hyperglycemia-induced ROS generation in podocytes. INO-1001 and PJ-34 also normalized the hyperglycemia-induced mitochondrial depolarization. PARP blockade by INO-1001 and PJ-34 prevented hyperglycemia-induced nuclear factor-kappaB (NFkappaB) activation of podocytes, and it was made evident by the inhibitor of kappaBalpha phosphorylation and NFkappaB p50 nuclear translocation. Our results indicate that hyperglycemia-induced PARP activation plays an important role in the pathogenesis of glomerulopathy associated with type 2 diabetes and could serve as a novel therapeutic target.

The Interaction of the Wnt and Notch Pathways Modulates NK vs. T Cell Commitment.

The Wnt and Notch signaling pathways have critical roles in cell fate decisions. However, the interaction of these pathways is poorly understood. Using highly purified Wnt3a and immobilized Notch ligand, Delta1ext-IgG, we investigated the mechanisms involved in Wnt and Notch signaling interactions during hematopoietic stem cell differentiation. When CD34+CD38- cord blood stem cells were cultured for 2 to 3 weeks with five growth factors (SCF 300ng/ml, Flt-3L 300ng/ml, TPO 100ng/ml, IL-6 100ng/ml, and IL-3 10ng/ml), most precursor cells lost CD34 expression and differentiated into mature cells, most of which were monocytes. However, as we previously reported, when cells were cultured with Delta1ext-IgG, we found an increased percentage of lymphoid progenitors (CD34+CD7+) and more mature lymphoid precursors (CD34-CD7+) in the cell population. The addition of purified Wnt3a (100ng/ml) alone without Delta1ext-IgG did not significantly change the percentage of CD7+ cells (1% vs. 3%). However, when both Wnt3a and Delta1ext-IgG were added, we saw an increased percentage of CD7+ cells (11% with Delta1ext-IgG alone to 27% with both) (Fig. 1A). Wnt3a also enhanced gene expression of CD3ε and preTα induced by Delta1ext-IgG. These results suggest that Wnt3a enhances the effect of Notch signaling on T-cell lineage development. To test the role of endogenous Wnt signaling, we added Dickkopf1, an inhibitor of Wnt signaling. When CD34+CD38- cells were cultured with Dickkopf1 (300ng/ml) alone for a 2 week, the percentage and number of CD56+ NK cells was unaffected. However, when cultured with Dickkopf1 and Delta1ext-IgG, the percentage and number of CD56+ NK precursor cells were increased (2% with Delta1ext-IgG alone vs. 17% with Dickkopf1 and Delta1ext-IgG; 0.2 ×105 vs. 1.5×105, p<0.01) (Fig. 1B). This result shows that decreased endogenous Wnt signaling enhances the generation of NK cells in the presence of Notch signaling. To address whether Wnt signaling affects Notch signaling by modulating protein levels, we assessed the amount of activated Notch1 intracellular domain with or without Wnt 3a by Western blot (Fig. 2A). After stimulation for 24 h, Wnt3a increased the amount of activated Notch1 intracellular domain induced by Delta1ext-IgG. Wnt3a also enhanced the expression of a primary target gene of Notch signaling, Hes1, determined by quantitative RT-PCR (Fig. 2B). In contrast, Dickkopf1 reduced Delta1ext-IgG-induced Hes1 expression. These results suggest that Wnt signaling directly modulates Notch signaling. Thus, these studies suggest that Wnt signaling is a key factor in cell fate determination at the point of NK/T cell commitment that is mediated via an interaction with Notch signaling. These studies also suggest that Wnt directly regulates Notch signaling by modulating protein turnover. [Display omitted]

Absolute Quantitation of Cancer-Related Proteins Using an MS-Based Peptide Chip

New technologies are needed that can diagnose cancer more rapidly and accurately. These technologies must also have the ability to identify the particular cellular abnormalities contributing to the malignancy, thus directing the appropriate treatments. Such technologies should permit absolute quantitation of specific tumor biomarkers and their level of posttranslational modifications. Quantitative molecular profiling of cancer signaling networks would provide a more detailed understanding of the contribution of protein expression and posttranslational modification levels to tumorigenesis. We have developed a unique approach for absolute quantitation of protein expression that integrates affinity capture of proteolytic peptides with mass spectrometry and thus provides detection, identification, and quantitation of their cognate proteins. We have previously shown the high sensitivity and specificity of this approach. Here we demonstrate the absolute quantitation of a model peptide using our technology. We have used this approach to capture epitope-containing peptides from proteolytically digested target proteins, including p53, epidermal growth factor receptor (EGFR), and prostate-specific antigen (PSA). Our technology can easily be extended to the absolute quantitation of protein modification levels, in addition to the determination of protein expression levels, and can be readily adapted for use in a microarray format. This method offers an improved approach to protein chip technology that should prove useful for clinical diagnosis and drug development applications.

Highly efficient modification of DNA polymerase β under conditions of direct and sensitized activation of photoreactive DNAs. Modification of cell extract proteins

dUTP and dCTP derivatives containing a 4-azido-2,3,5,6-tetrafluorobenzylideneaminooxy group were incorporated into the 3′-end of the DNA primer within complexes with the DNA-matrix as analogs of natural dTTP by virtue of catalytic activity of DNA polymerase β or endogenous DNA polymerases of the cell extract. The photoreactive DNAs synthesized in situ were used for affinity modification of DNA polymerase β and DNA-binding proteins of the cell extract. For the photoreactive DNA based on these analogs, the efficiency of formation of covalent adducts with DNA polymerase β under the highest degree of DNA complexation with the enzyme was determined. The yield of covalent DNA adducts with the enzyme was 28–47%, depending on the type of the analog. The effect of the sequence of the DNA template near the localization of the photoreactive group on the redistribution of covalent cross-links between the possible targets was demonstrated. A possibility of increasing the efficiency of DNA polymerase β modification in the presence of a substantial excess of photoreactive DNA using a sensitizer, a dUTP derivative containing a pyrene residue, was studied. When photoreactive DNA containing a 2,3,5,6-tetrafluoro-4-azidobenzoyl (FAB) group was used, about 60% of DNA polymerase β was covalently attached to DNA. Photoreactive dNTP analogs ensuring a high level of protein modification in the cell extract were found.

In vivo and in vitro oxidative regulation of rat aryl sulfotransferase IV (AST IV)

Sulfotransferase catalyzed sulfation is important in the regulation of different hormones and the metabolism of hydroxyl containing xenobiotics. In the present investigation, we examined the effects of hyperoxia on aryl sulfotransferase IV in rat lungs in vivo. The enzyme activity of aryl sulfotransferase IV increased 3- to 8-fold in >95% O2 treated rat lungs. However, hyperoxic exposure did not change the mRNA and protein levels of aryl sulfotransferase IV in lungs as revealed by Western blot and RT-PCR. This suggests that oxidative regulation occurs at the level of protein modification. The increase of nonprotein soluble thiol and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios in treated lung cytosols correlated well with the aryl sulfotransferase IV activity increase. In vitro, rat liver cytosol 2-naphthol sulfation activity was activated by GSH and inactivated by GSSG. Our results suggest that Cys residue chemical modification is responsible for the in vivo and in vitro oxidative regulation. The molecular modeling structure of aryl sulfotransferase IV supports this conclusion. Our gel filtration chromatography results demonstrated that neither GSH nor GSSG treatment changed the existing aryl sulfotransferase IV dimer status in cytosol, suggesting that oxidative regulation of aryl sulfotransferase IV is not caused by dimer-monomer status change.

Tyrosine nitration in plasma proteins from patients undergoing hemodialysis

Background: A growing body of evidence suggesting that oxidative stress might be one of the most important complications occurring during hemodialysis (HD) has accumulated. However, although the role of reactive oxygen species has been investigated extensively, little is known about the involvement of reactive nitrogen species. In the present investigation, levels of protein modifications in the form of tyrosine nitration in patients undergoing long-term HD therapy were evaluated. Methods: Tyrosine nitration of plasma proteins was detected by means of Western blotting using a specific nitrotyrosine-recognizing monoclonal antibody, and band intensity was evaluated by using image analysis software. Immunoprecipitation of plasma proteins by antinitrotyrosine-agarose—conjugated antibodies, followed by Western blotting, was used in an attempt to identify the nitrated proteins. Results: Although several proteins with nitrated tyrosine residues were observed in plasma of healthy individuals, increased nitration levels were observed in some specific proteins in all patients tested (n = 25) compared with controls (n = 6). At least 6 apparent bands appeared to be more nitrated than their counterparts in plasma from controls. Ceruloplasmin was identified as 1 of the proteins with significantly increased nitration in patients. Conclusion: Results of the present investigation show that specific plasma proteins of HD patients are post-translationally modified by nitration of their tyrosine residues. The nature of these proteins, as well as the exact molecular mechanisms and consequences of these modifications, warrant additional investigation.

Exploring the functional complexity of cellular proteins by protein knockout.

Comprehensive dissection of protein functions entails more complicated manipulations than simply eliminating the protein of interest. Established knockdown technologies, such as RNA interference, antisense oligodeoxynucleotides, or ribozymes, are limited for specific applications such as modulating protein levels or specific targeting of a posttranslationally modified subpopulation. Here we show that the engineered Skp1, Cullin 1, and F-box-containing betaTrCP substrate receptor ubiquitin-proteolytic system, designated protein knockout, could achieve not only total elimination but also rapid and systematic reduction of a given cellular protein. Stable expression of a single engineered betaTrCP demonstrated simultaneous and sustained degradation of the entire retinoblastoma family proteins. Furthermore, the engineered betaTrCP was capable of selecting hypo- but not hyperphosphorylated forms of retinoblastoma for degradation. The engineered betaTrCP has been extensively modified to increase its specificity in substrate selection. This optimized protein-knockout system offers a powerful and versatile proteomic tool to dissect diverse functional properties of cellular proteins in somatic cells.

Protein oxidation in aging: endoplasmic reticulum as a target.

Oxidatively modified proteins have been shown to correlate with the age of an organism or its tissues. An increase in tissue-susceptibility to experimentally induced protein oxidation not only depends on tissue type and age, but also on the maximum lifespan potential of the species. A general, although tissue dependent, decline in anti-oxidative defenses during aging may very well be responsible for this difference in vulnerability. In addition, the level of protein modifications also depends on the nature and the subcellular localization of the proteins involved. Damage to the endoplasmic reticulum (ER), and its subsequent impaired functionality may be involved in the process of aging. This is suggested by; (1) an upregulation of ER stress-response chaperones, (2) a preferential oxidation of ER-resident proteins and, (3) a disturbance of calcium homeostasis. Therefore, this review will focus on the putative involvement of the oxidized endoplasmic reticulum in the process of aging.

The cell cycle and seed germination

AbstractThe cell cycle is the series of molecular events that allows cells to duplicate and segregate their chromosomes to form new cells. The finding that a protein kinase, the product of the yeastcdc2gene, was fundamental in the regulation of the G2/M and G1/S transitions, associated with unstable proteins named cyclins, opened a very exciting and dynamic research area. The number of gene products that participate in the development and regulation of the cell cycle may be in the hundreds, and there is a high degree of conservation in protein sequences and regulatory pathways among eukaryotes. Although there are clear differences between plants and animals in cell structure, organization, growth, development and differentiation, the same types of proteins and very similar regulatory pathways seem to exist. Seed germination appears to be an excellent model system for studying the cell cycle in plants. Imbibition will reactivate meristematic cells – most initially with a G1DNA content – into the cell cycle in preparation for seedling establishment. Early events include a thorough survey of DNA status, since the drying process and seed storage conditions reduce chromosomal integrity. The initiation of cell cycle events leading to G1and S phases, and of the germination process itself, may depend on a G1checkpoint control. Most, if not all, cell cycle proteins appear to be already present in unimbibed embryos, although there is evidence of protein turnover in the early hours, suggesting the need forde novoprotein synthesis. Regulation also may occur at the level of protein modification, because existing G1, S and G2cell cycle proteins appear to be activated at precise times during germination. Thus, cell cycle control during seed germination may be exerted at multiple levels; however, knowledge of cell cycle events and their importance for germination is still scarce and fragmentary, and different species may have developed unique control mechanisms, more suited to specific germination characteristics and habitat.

Variants of peroxiredoxins expression in response to hydroperoxide stress

We examined patterns of the proteins that were expressed in human umbilical vein endothelial cells (HUVEC) in response to oxidative stress by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). When HUVEC were exposed to H 2O 2 at 100 μM for 60 min, the intensities of eight spots increased and those of eight spots decreased on 2D gels, as compared with control gels, after staining with silver. These changes were also observed after exposure of cells to hydroperoxides such as cumene hydroperoxide and tert-butyl hydroperoxide, but not after exposure to other reagents that induce oxidative stress such as S-alkylating compounds, nitric oxide, and salts of heavy metals. Therefore, these proteins were designated hydroperoxide responsive proteins (HPRPs). Microsequencing analysis revealed that these HPRPs corresponded to at least six pairs of proteins. Of these, four pairs of HPRPs were thioredoxin peroxidase I (TPx I), TPx II, TPx III, and the product of human ORF06, all of which belong to the peroxiredoxin (Prx) family and all of which are involved in the elimination of hydroperoxides. The other two pairs corresponded to heat shock protein 27 (HSP27) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH), respectively. The variants that appeared in response to hydroperoxides had molecular masses similar to the respective native forms, but their pI values were lower by 0.2–0.3 pH units than those of the corresponding native proteins. These variants were detected on 2D gels after cells had been exposed to hydroperoxides in the presence of an inhibitor of protein synthesis. All variants were generated within 30 min of exposure to 100 μM H 2O 2. The variants of TPx I and TPx II appeared within 2 min of the addition of H 2O 2 to the culture medium. The HPRPs returned to their respective native forms after the removal of stress. Our results indicated that at least six proteins were structurally modified in response to hydroperoxides. Analysis by 2D-PAGE of 32P-labeled proteins revealed that the variant of HSP27 was its phosphorylated form while the other HPRPs were not modified by phosphorylation. Taken together, the results suggest that 2D-PAGE can reveal initial responses to hydroperoxide stress at the level of protein modification. Moreover, it is possible that the variants of four types of Prx might reflect intermediate states in the process of hydroperoxide elimination.

RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination.

A RING finger-containing protein (AO7) that binds ubiquitin-conjugating enzymes (E2s) and is a substrate for E2-dependent ubiquitination was identified. Mutations of cation-coordinating residues within AO7's RING finger abolished ubiquitination, as did chelation of zinc. Several otherwise-unrelated RING finger proteins, including BRCA1, Siah-1, TRC8, NF-X1, kf-1, and Praja1, were assessed for their ability to facilitate E2-dependent ubiquitination. In all cases, ubiquitination was observed. The RING fingers were implicated directly in this activity through mutations of metal-coordinating residues or chelation of zinc. These findings suggest that a large number of RING finger-containing proteins, with otherwise diverse structures and functions, may play previously unappreciated roles in modulating protein levels via ubiquitination.

Introduction: antisense oligonucleotides: strategies and successes.

New approaches to examining the nervous system are applicable to studies of the chemical senses. Various technical interventions have been developed to alter or 'delete' a gene product to provide insight into the role of a particular macromolecule during development, during cell-cell signaling or in a behavioral context. Antisense oligodeoxynucleotide (ODN) delivery to the cell nucleus prevents translation of an mRNA of interest, thus eliminating the protein product from the cell. This approach offers certain advantages over other technologies that modify gene products. In theory, expression of proteins of interest may be decreased at will at any time by introduction of an antisense ODN that interferes with synthesis of the protein from its mRNA. In its optimal embodiment, antisense 'knockout' of a protein through eliminating its cognate mRNA is reversible, and the strategy could be used in a range of species at any time during the development or life of the animal. At the desired time point, gene blockade could be tested for its effects on development, on responses to particular signals, or on learning or behavior before or after a training paradigm. This approach circumvents some of the disadvantages of other technologies that modify protein levels. For example, transgenic techniques have been widely utilized to introduce new genes that result in a 'knockout' or 'knock-in' animal. Genes are deleted or replaced by swapping new genetic material for an endogenous gene through homologous recombination events (van der Neut, 1997). The new construct may delete the reading sequence of the target gene or contain a stop signal to eliminate translation into protein, or produce a truncated, non-functional protein product. However, production of 'knockouts' is technically demanding, time-consuming and expensive. Results are sometimes disappointing, yielding deficiencies that are lethal to the embryo so that no viable animals are born. Further, gene deletion sometimes appears to have no apparent consequences, perhaps because the protein product is redundant or because alternative pathways activated during development replace the function of the missing gene or increase expression of a compensatory gene(s).

Proteolysis in plants: mechanisms and functions.

Proteolysis is essential for many aspects of plant physiology and development. It is responsible for cellular housekeeping and the stress response by removing abnormal/misfolded proteins, for supplying amino acids needed to make new proteins, for assisting in the maturation of zymogens and peptide hormones by limited cleavages, for controlling metabolism, homeosis, and development by reducing the abundance of key enzymes and regulatory proteins, and for the programmed cell death of specific plant organs or cells. It also has potential biotechnological ramifications in attempts to improve crop plants by modifying protein levels. Accumulating evidence indicates that protein degradation in plants is a complex process involving a multitude of proteolytic pathways with each cellular compartment likely to have one or more. Many of these have homologous pathways in bacteria and animals. Examples include the chloroplast ClpAP protease, vacuolar cathepsins, the KEX2-like proteases of the secretory system, and the ubiquitin/26S proteasome system in the nucleus and cytoplasm. The ubiquitin-dependent pathway requires that proteins targeted for degradation become conjugated with chains of multiple ubiquitins; these chains then serve as recognition signals for selective degradation by the 26S proteasome, a 1.5 MDa multisubunit protease complex. The ubiquitin pathway is particularly important for developmental regulation by selectively removing various cell-cycle effectors, transcription factors, and cell receptors such as phytochrome A. From insights into this and other proteolytic pathways, the use of phosphorylation/dephosphorylation and/or the addition of amino acid tags to selectively mark proteins for degradation have become recurring themes.