Cytoskeleton changes following differentiation of N1E-115 neuroblastoma cell line

Summary. No systematic approach to detect expression of differentiation-related elements was published so far. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments. We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical identification of proteins to generate a map of cytoskeleton proteins (CPs), i.e., to search for differentiation-related structures. Alpha-actin, actin-like protein 6A, gamma-tubulin complex component 2, tubulin alpha 3/alpha 7, CLIP associating protein 2, B4 integrin interactor homolog were detectable in the undifferentiated cell line exclusively and neuron-specific CPs drebrin and presynaptic density protein 95, actin-related protein 2/3, alpha and beta-centractin, PDZ-domain actin binding protein, actinin alpha 1, profilin II, ezrin, coactosin-like protein, transgelin 2, myosin light polypeptide 6, tubulin alpha 2, 6 and 7, beta tubulin (94% similar with tubulin beta-2), tubulin beta 3, tubulin tyrosine ligase-like protein 1, lamin B1 and keratin 20 were observed in the differentiated cell line only. We herein identified differentiation-related expressional patterns thus providing new evidence for the role of CPs in the process of neuronal differentiation.     

Eye lens proteomics

Summary. The eye lens is a fascinating organ as it is in essence living transparent matter. Lenticular transparency is achieved through the peculiarities of lens morphology, a semi-apoptotic process where cells elongate and loose their organelles and the precise molecular arrangement of the bulk of soluble lenticular proteins, the crystallins. The 16 crystallins ubiquitous in mammals and their modifications have been extensively characterized by 2-DE, liquid chromatography, mass spectrometry and other protein analysis techniques. The various solubility dependant fractions as well as subproteomes of lenticular morphological sections have also been explored in detail. Extensive post translational modification of the crystallins is encountered throughout the lens as a result of ageing and disease resulting in a vast number of protein species. Proteomics methodology is therefore ideal to further comprehensive understanding of this organ and the factors involved in cataractogenesis.     

Translocation and induction of alpha B crystallin by heat shock in rat glioma (GA-1) cells.

Response to heat shock of alpha B crystallin expressed in rat astrocytoma GA-1 cells was analysed quantitatively using an immunoassay method. GA-1 cells contained a considerable amount of alpha B crystallin at growing phase. When the growing cells were heated at 45 degrees C, concentrations of alpha B crystallin in cell extracts were decreased to less than one-fifth of the original level within 15 min, with an increase in the insoluble fraction which was detected by immunoblotting. The low level of alpha B crystallin in the cytoplasm, that was observed for a few hours after heat shock, gradually recovered to the control level within several hours. At 10 h following heat shock (45 degrees C for 15 min), the concentration of alpha B crystallin in the soluble extract was about twice that of the control level, with little detectable amounts in the insoluble fraction. These results are additional evidence that suggest that alpha B crystallin is a small heat shock protein.     

Sulfenic acid in human serum albumin

Summary. Sulfenic acid (RSOH) is a central intermediate in both the reversible and irreversible redox modulation by reactive species of an increasing number of proteins involved in signal transduction and enzymatic pathways. In this paper we focus on human serum albumin (HSA), the most abundant plasma protein, proposed to serve antioxidant functions in the vascular compartment. Sulfenic acid in HSA has been previously detected using different methods after oxidation of its single free thiol Cys34 through one- or two-electron mechanisms. Since recent evidence suggests that sulfenic acid in HSA is stabilized within the protein environment, this derivative represents an appropriate model to examine protein sulfenic acid biochemistry, structure and reactivity. Sulfenic acid in HSA could be involved in mixed disufide formation, supporting a role of HSA-Cys34 as an important redox regulator in extracellular compartments.     

Spermine and spermidine mediate protection against oxidative damage caused by hydrogen peroxide

Summary. The polyamines spermidine and spermine have been hypothesized to possess different functions in the protection of DNA from reactive oxygen species. The growth and survival of mouse fibroblasts unable to synthesize spermine were compared to their normal counterparts in their native and polyamine-depleted states in response to oxidative stress. The results of these studies suggest that when present at normal or supraphysiological concentrations, either spermidine or spermine can protect cells from reactive oxygen species. However, when polyamine pools are pharmacologically manipulated to produce cells with low levels of predominately spermine or spermidine, spermine appears to be more effective. Importantly, when cells are depleted of both glutathione and endogenous polyamines, they exhibit increased sensitivity to hydrogen peroxide as compared to glutathione depletion alone, suggesting that polyamines not only play a role in protecting cells from oxidative stress but this role is distinct from that played by glutathione.     

Age-related changes of alpha-crystallin aggregate in human lens

Summary. Lens alpha-crystallin, composed of two subunits alpha A- and alpha B-crystallin, forms large aggregates in the lens of the eye. The present study investigated the aggregate of human lens alpha-crystallin from elderly and young donors. Recombinant alpha A- and alpha B-crystallins in molar ratios of alpha A to alpha B at 1:1, corresponding to the aged sample, were also studied in detail. We found by ultra-centrifugation analysis that the alpha-crystallin aggregate from elderly donors was large and heterogeneous with an average sedimentation coefficient of 30 S and a range of 20–60 S at 37 °C. This was higher compared to the young samples that had an average sedimentation coefficient of 17 S. The sedimentation coefficients of recombinant alpha A- and alpha B-crystallins were approximately 12 S and 15 S, respectively. Even when recombinant alpha-crystallins were mixed in molar ratios equivalent to those found in vivo, similar S values as the native aged alpha-crystallin aggregates were not obtained. Changes in the self-association of alpha-crystallin aggregate were correlated to changes in chaperone activity. Alpha-crystallin from young donors, and recombinant alpha A- and alpha B-crystallin and their mixtures showed chaperone activity, which was markedly lost in samples from the aged alpha-crystallin aggregates.     

Kinetic and proteomic analyses of <Emphasis Type="Italic">S</Emphasis>-nitrosoglutathione-treated hexokinase A: consequences for cancer energy metabolism

Summary. Mammalian hexokinase (HXK) is found at the outer mitochondrial membrane, exposed to mitochondrial oxygen- and nitrogen-radicals. Given the important role of this enzyme in metabolic pathways and diseases, the effect of S-nitrosoglutathione (GSNO) on HXK A structure and activity was studied. To focus on the catalytic domain, yeast HXK A was used because it has a significant homology to the mammalian domain that contains both the regulatory and catalytic sites. Biologically relevant [GSNO]/[HXK] caused a significant decrease in V_max with glucose (but not with fructose), along with oxidation of 5 Met and nitration of 4 Tyr. Preincubation of HXK with glucose abrogated the effect of GSNO whereas fructose was ineffective. These results are interpreted by considering the tight binding of glucose to the enzyme as opposed to that of fructose. The segment comprised from amino acids 304 to 306 contained the most modifications. Given that this sequence is highly conserved in HXK from various species, a decline in activity is expected when a high-affinity substrate is presented. Considering that changes in primary structure are envisioned at high [GSNO]/[HXK] ratios, like those present under normal conditions, it could be hypothesized that the high concentration of hexokinase present in fast growing tumors may serve not only to sustain high glycolysis rates, but also to minimize protein damage that might result in activity decline, compromising energy metabolism.     

Effect of cycloheximide on tryptophan binding to rat hepatic nuclei

Summary. This study evaluated whether cycloheximide, an inhibitor of protein synthesis, would affect the binding of L-tryptophan to rat hepatic nuclei or nuclear envelopes. Previous reports have indicated that the binding of L-tryptophan to hepatic nuclear envelope protein was saturable, stereospecific, and of high affinity. Also, the administration of L-tryptophan rapidly stimulated hepatic protein synthesis. In this study, we determined that the addition of cycloheximide in vitro inhibited ³H-tryptophan binding to hepatic nuclei or nuclear envelopes. Heat-treated cycloheximide failed to have this inhibitory binding effect. In vivo treatment of rats with cycloheximide diminished in vitro ³H-tryptophan binding to hepatic nuclei of treated rats compared to controls. Puromycin, another inhibitor of hepatic protein synthesis, when added in vitro did not affect ³H-tryptophan binding to hepatic nuclei but did diminish in vitro binding after in vivo treatment. Thus, cycloheximide added in vitro diminished ³H-tryptophan binding to hepatic nuclei probably by its structural effect on the receptor while cycloheximide administered in vivo may also act in part by inhibiting protein synthesis. Received March 22, 1999, Accepted May 31, 1999     

Regulation and activation of phytoene synthase, a key enzyme in carotenoid biosynthesis, during photomorphogenesis

 During photomorphogenesis in higher plants, a coordinated increase occurs in the chlorophyll and carotenoid contents. The carotenoid level is under phytochrome control, as reflected by the light regulation of the mRNA level of phytoene synthase (PSY), the first enzyme in the carotenoid biosynthetic pathway. We investigated PSY protein levels, enzymatic activity and topological localization during photomorphogenesis. The results revealed that PSY protein levels and enzymatic activity increase during de-etiolation and that the enzyme is localized at thylakoid membranes in mature chloroplasts. However, under certain light conditions (e.g., far-red light) the increases in PSY mRNA and protein levels are not accompanied by an increase in enzymatic activity. Under those conditions, PSY is localized in the prolamellar body fraction in a mostly enzymatically inactive form. Subsequent illumination of dark-grown and/or in far-red light grown seedlings with white light causes the decay of these structures and a topological relocalization of PSY to developing thylakoids which results in its enzymatic activation. This light-dependent mechanism of enzymatic activation of PSY in carotenoid biosynthesis shares common features with the regulation of the NADPH:protochlorophyllide oxidoreductase, the first light-regulated enzyme in chlorophyll biosynthesis. The mechanism of regulation described here may contribute to ensuring a spatially and temporally coordinated increase in both carotenoid and chlorophyll contents. Received: 14 February 2000 / Accepted: 15 March 2000     

Hallmarks of protein oxidative damage in neurodegenerative diseases: focus on Alzheimer’s disease

Summary. The pathogenesis of several neurodegenerative diseases, including Alzheimer’s disease, has been linked to a condition of oxidative and nitrosative stress, arising from the imbalance between increased reactive oxygen species (ROS) and reactive nitrogen species (RNS) production and antioxidant defences or efficiency of repair or removal systems. The effects of free radicals are expressed by the accumulation of oxidative damage to biomolecules: nucleic acids, lipids and proteins. In this review we focused our attention on the large body of evidence of oxidative damage to protein in Alzheimer’s disease brain and peripheral cells as well as in their role in signalling pathways. The progress in the understanding of the molecular alterations underlying Alzheimer’s disease will be useful in developing successful preventive and therapeutic strategies, since available drugs can only temporarily stabilize the disease, but are not able to block the neurodegenerative process.     

Ubiquitin-protein conjugates and alpha B crystallin are selectively present in cells undergoing major cytomorphological reorganisation in early chicken embryos.

Ubiquitin-protein conjugates and alpha B crystallin are detected immunohistochemically in cells undergoing extensive morphological reorganisation in early chicken embryos. Cytoplasmic ubiquitinated proteins and alpha B crystallin are coordinately found in cells of the lens, notochord and myotome. The antigens appear in the myotome cells precisely at the point at which the cells begin to migrate from the dorsomedial lip of the dermamyotome. The findings indicate that ubiquitin and alpha B crystallin may have a coordinate role in the extensive architectural remodeling which occurs in these developing tissues in the early chick embryo. Some form of functional association between protein ubiquitination and alpha B crystallin in cells may explain why alpha B crystallin is found with ubiquitin-protein deposits in some neurodegenerative diseases.     

Evolution of crystallins: expression of lens-specific proteins in the blind mammals mole (Talpa europaea) and mole rat (Spalax ehrenbergi)

The mole (Talpa europaea; Insectivora) and the mole rat (Spalax ehrenbergi; Rodentia) both have degenerated eyes as a convergent adaptation to subterranean life. The rudimentary eye lenses of these blind mammals no longer function in a visual process. The crystallin genes, which display a lens-specific expression pattern, were studied in these blind mammals and in related species with normal eyes by hybridizing their genomic DNAs with probes obtained from cDNA clones for alpha A-, alpha B-, and beta Bp-crystallins from calf and gamma 3- crystallin from the rat. For all crystallin genes examined, the hybridization signals of mole and mole rat genomic DNA were comparable, respectively, with those of shrew and of rat and mouse, normal-vision representatives of the orders Insectivora and Rodentia. The expression of the crystallins at the protein level was tested by using antiserum specific for alpha-crystallin in immunofluorescence reactions on lens sections of mole and mole rat eyes and by using antisera against the beta- and gamma-crystallins on sections of the mole eye. All antisera gave positive fluorescence reactions exclusively with lens tissue of these blind mammals, indicating that the crystallins are still normally expressed despite the fact that these lenses have had no function in a visual process in these mammals for at least many million years. These findings apparently imply that some unknown selective advantage has conserved the crystallin genes and their expression after the loss of normal function of the lenses.      

Lysine catabolism, an effective versatile regulator of lysine level in plants

Lysine is a nutritionally important essential amino acid, whose synthesis in plants is strongly regulated by the rate of its synthesis. Yet, lysine level in plants is also finely controlled by a super-regulated catabolic pathway that catabolizes lysine into glutamate and acetyl Co-A. The first two enzymes of lysine catabolism are synthesized from a single LKR/SDH gene. Expression of this gene is subject to compound developmental, hormonal and stress-associated regulation. Moreover, the LKR/SDH gene of different plant species encodes up to three distinct polypeptides: (i) a bifunctional enzyme containing the linked lysine-ketoglutarate (LKR) and saccharopine dehydrogenase (SDH) whose LKR activity is regulated by its linked SDH enzyme; (ii) a monofunctional SDH encoded by an internal promoter, which is a part of the coding DNA region of the LKR/SDH gene; and (iii) a monofunctional, highly potent LKR that is formed by polyadenylation within an intron. LKR activity in the bifunctional LKR/SDH polypeptide is also post-translationally regulated by phosphorylation by casein kinase-2 (CK2), but the consequence of this regulation is still unknown. Why is lysine metabolism super-regulated by synthesis and catabolism? A hypothesis addressing this important question is presented, suggesting that lysine may serve as a regulator of plant growth and interaction with the environment.     

Studies of alphaB crystallin subunit dynamics by surface plasmon resonance

The molecular chaperone activity of alphaB crystallin, an important stress protein in humans, is regulated by physiological factors, including temperature, pH, Ca2+, and ATP. In this study, the role of these factors in regulating the subunit dynamics of human alphaB crystallin was investigated using surface plasmon resonance (SPR). SPR experiments indicate that at temperatures above 37 degrees C, where alphaB crystallin has been reported to have higher chaperone activity, the subunit dynamics of alphaB crystallin were increased with faster association and dissociation rates. SPR experiments also indicate that interactions between alphaB crystallin subunits were enhanced with much faster association and slower dissociation rates at pH values below 7.0, where alphaB crystallin has been reported to have lower chaperone activity. The results suggest that the dynamic and rapid subunit exchange rate may regulate the chaperone activity of alphaB crystallin. The effect of Ca2+ and ATP on the subunit dynamics of alphaB crystallin was minimal, suggesting that Ca2+ and ATP modulate the chaperone activity of alphaB crystallin without altering the subunit dynamics. Based on the SPR results and previously reported biochemical data for the chaperone activity of alphaB crystallin under different conditions of temperature and pH, a model for the relationship between the subunit dynamics and chaperone activity of alphaB crystallin is established. The model is consistent with previous biochemical data for the chaperone activity and subunit dynamics of small heat shock proteins (sHSPs) and establishes a working hypothesis for the relationship between complex assembly and chaperone activity for sHSPs.     

Peptide mass fingerprinting

Peptide mass fingerprinting by MALDI-MS and sequencing by tandem mass spectrometry have evolved into the major methods for identification of proteins following separation by two-dimensional gel electrophoresis, SDS-PAGE or liquid chromatography. One main technological goal of proteome analyses beside high sensitivity and automation was the comprehensive analysis of proteins. Therefore, the protein species level with the essential information on co- and post-translational modifications must be achieved. The power of peptide mass fingerprinting for protein identification was described here, as exemplified by the identification of protein species with high molecular masses (spectrin alpha and beta), low molecular masses (elongation factor EF-TU fragments), splice variants (alpha A crystallin), aggregates with disulfide bridges (alkylhydroperoxide reductase), and phosphorylated proteins (heat shock protein 27). Helpful tools for these analyses were the use of the minimal protein identifier concept and the software program MS-Screener to remove mass peaks assignable to contaminants and neighbor spots.     

Recombinant human alpha-1 proteinase inhibitor: towards therapeutic use

Summary. Human alpha-1-proteinase inhibitor is a well-characterized protease inhibitor with a wide spectrum of anti-protease activity. Its major physiological role is inhibition of neutrophil elastase in the lungs, and its deficiency is associated with progressive ultimately fatal emphysema. Currently in the US, only plasma-derived human alpha-1-proteinase inhibitor is available for augmentation therapy, which appears to be insufficient to meet the anticipated clinical demand. Moreover, despite effective viral clearance steps in the manufacturing process, the potential risk of contamination with new and unknown pathogens still exists. In response, multiple efforts to develop recombinant versions of human alpha-1-proteinase inhibitor, as an alternative to the plasma-derived protein, have been reported. Over the last two decades, various systems have been used to express the human gene for alpha-1-proteinase inhibitor. This paper reviews the recombinant versions of human alpha-1-proteinase inhibitor produced in various hosts, considers current major safety and efficacy issues regarding recombinant glycoproteins as potential therapeutics, and the factors that are impeding progress in this area¹. ¹ The opinions expressed in this paper reflect the authors’ personal views, based on published data and the information available from the public domains, and have no relation to the official statements, if any, held by the US FDA, National Institutes of Health, or the US Department of Health and Human Services. FDA official recommendations for plasma protein therapeutics and recombinant proteins regarding safety, purity, and potency of new drugs and biologics produced by recombinant DNA technology are referred below as the US FDA Guidances.     

Copurification of small heat shock protein with alpha B crystallin from human skeletal muscle.

Immunoreactive alpha B crystallin and a 28-kDa protein in an extract of human pectoral muscle were precipitated by (NH4)2SO4 at 40% saturation, and coeluted during column chromatography on DEAE-Sepharose and on Bio-Gel A-5m. The two proteins were separated on a column of S-Sepharose HP in the presence of 7 M urea. Further chromatography of each of the two resultant fractions on a column of Superdex 75 pg and on a TSK-SP 5PW column in the presence of urea yielded preparations of alpha B crystallin and the 28-kDa protein each of which gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final preparation of 28-kDa protein contained at least two subtypes, which were separable on the TSK-SP column. However, fragmentation patterns of the two major 28-kDa proteins after digestion with endoproteinase Asp-N were identical. Amino acid sequences of peptides formed by cleavage of the purified 28-kDa protein and alpha B crystallin were identical to those of particular regions of the deduced amino acid sequences of human small heat shock protein (HSP28) and lens alpha B crystallin, respectively. Using an immunoassay method, with antibodies raised in rabbits, we found that HSP28 was present in all human tissues tested and at high levels (greater than 1 micrograms/mg protein) in the heart and other tissues composed of striated and smooth muscles. HSP28, found with alpha B crystallin, in extracts of several human and bovine tissues was trapped on and coeluted with alpha B crystallin from an affinity column prepared with antibodies against alpha B crystallin. This result suggests that the two proteins are associated in cells.