Metabolic behaviors of the core histones in proliferating Friend cells

This study examines the turnover of the core histones in proliferating Friend cells. It was calculated that these proteins turn over with half-lives of 21.6 days for H2A, 13.8 days for H2B, 43.3 days for H3, and 138.6 days for H4. The significant differences in the half-lives of the four core histones indicate that the protein moiety of the nucleosome is not replaced as one entire unit but as a "mosaic" in which each component follows its own rate of replacement. In some experiments the turnover rates of the variants of H2A, H2B, and H3 were compared. The results did not indicate any differences among these histone variants, suggesting that they are not excluded from the mechanisms controlling histone turnover. Metabolic heterogeneity was discovered, however, when the turnover rates of the acetylated and nonacetylated molecules of histone H4 were followed: it appeared that the acetylated molecules are replaced 2.5 times faster. The comparison of the rate of replacement of the histones in proliferating and differentiated cells from one site and their level of acetylation from another suggests that this postsynthetic modification might be involved in the control of histone metabolism. Such a conclusion is supported also by a number of model experiments.     

Structure, biosynthesis, and function of the hexose transporter in Chinese hamster ovary cells deficient in N-acetylglucosaminyltransferase 1 activity

We have used a Chinese hamster ovary cell line deficient in N-acetylglucosaminyltransferase 1 activity (Lec1) to study the effects of altered asparagine-linked oligosaccharides on the structure, biosynthesis, and function of glucose transporter protein. Immunoblots of membranes of Lec1 cells show a glucose transporter protein of Mr 40,000, whereas membranes of wild-type (WT) cells contain a broadly migrating Mr 55,000 form similar to that observed in several other mammalian tissues. The total content of immunoreactive glucose transporters in Lec1 cells is 3.5-fold greater than that of WT cells. Digestion with endoglycosidases, treatment with inhibitors of glycosylation, and interactions with agarose-bound lectins demonstrate that glucose transporters of both cell lines derive from a similar Mr 38,000 core polypeptide and that both contain asparagine-linked oligosaccharide. Transporters in Lec1 cells contain primarily "undecorated" but "trimmed" mannose-type asparagine-linked oligosaccharides, while the protein in WT cells contains a mixture of "decorated" and "trimmed" asparagine-linked oligosaccharides. Biosynthetic and turnover studies demonstrate that Lec1 cells, in contrast to WT cells, are unable fully to process the core asparagine-linked oligosaccharides of maturing glucose transporters. When radiolabeled in methionine-deficient medium both Lec1 and WT cells show similar rates of synthesis and turnover of glucose transporter proteins. It should be noted, however, that starvation for a critical amino acid may alter the ability of the cell to synthesize or degrade proteins. The abilities of Lec1 and WT cells to transport hexoses and to interact with the inhibitor cytochalasin B are very similar. The results indicate that, although altered asparagine-linked glycosylation can affect the content and biogenesis of glucose transporters, these changes do not greatly modify cellular hexose uptake. The possibility that alterations in asparagine-linked glycosylation may change the cell surface localization or acquisition of a "functional conformation" of the glucose transporter is also suggested.     

Regulation of intestinal protein metabolism by amino acids

Gut homeostasis plays a major role in health and may be regulated by quantitative and qualitative food intake. In the intestinal mucosa, an intense renewal of proteins occurs, at approximately 50 % per day in humans. In some pathophysiological conditions, protein turnover is altered and may contribute to intestinal or systemic diseases. Amino acids are key effectors of gut protein turnover, both as constituents of proteins and as regulatory molecules limiting intestinal injury and maintaining intestinal functions. Many studies have focused on two amino acids: glutamine, known as the preferential substrate of rapidly dividing cells, and arginine, another conditionally essential amino acid. The effects of glutamine and arginine on protein synthesis appear to be model and condition dependent, as are the involved signaling pathways. The regulation of gut protein degradation by amino acids has been minimally documented until now. This review will examine recent data, helping to better understand how amino acids regulate intestinal protein metabolism, and will explore perspectives for future studies.     

Enzymic nature of the protein moiety of protochlorophyllide holochrome

The enzymic nature of the protein moiety of protochlorophyll(ide) holochrome was studied by following the fate of the [(14)C]protochlorophyll(ide) formed when dark-grown barley (Hordeum vulgare) or bean (Phaseolus vulgaris) leaves are incubated in the dark with 3 mm 4-delta-[(14)C]aminolevulinic acid. It was found that: [List: see text]Since turnover of protochlorophyll(ide) was not observed, these results show that there is a free exchange between the old "endogenous" and the new delta-aminolevulinic-acid-induced protochlorophyll(ide) molecules on the active site of the holochrome protein. These results are consistent with the hypothesis that the holochrome protein acts as an enzyme.     

A cell based screening approach for identifying protein degradation regulators

Cellular transitions are achieved by the concerted actions of regulated degradation pathways. In the case of the cell cycle, ubiquitin mediated degradation ensures unidirectional transition from one phase to another. For instance, turnover of the cell cycle regulator cyclin B1 occurs after metaphase to induce mitotic exit. To better understand pathways controlling cyclin B1 turnover, the N-terminal domain of cyclin B1 was fused to luciferase to generate an N-cyclin B1-luciferase protein that can be used as a reporter for protein turnover. Prior studies demonstrated that cell-based screens using this reporter identified small molecules inhibiting the ubiquitin ligase controlling cyclin B1-turnover. Our group adapted this approach for the G2-M regulator Wee1 where a Wee1-luciferase construct was used to identify selective small molecules inhibiting an upstream kinase that controls Wee1 turnover. In the present study we present a screening approach where cell cycle regulators are fused to luciferase and overexpressed with cDNAs to identify specific regulators of protein turnover. We overexpressed approximately 14,000 cDNAs with the N-cyclin B1-luciferase fusion protein and determined its steady-state level relative to other luciferase fusion proteins. We identified the known APC/C regulator Cdh1 and the F-box protein Fbxl15 as specific modulators of N-cyclin B1-luciferase steady-state levels and turnover. Collectively, our studies suggest that analyzing the steady-state levels of luciferase fusion proteins in parallel facilitates identification of specific regulators of protein turnover.     

Mechanisms of protein degradation in mantle muscle and proposed gill remodeling in starved Sepia officinalis

Cephalopods have relatively high rates of protein synthesis compared to rates of protein degradation, along with minimal carbohydrate and lipid reserves. During food deprivation on board protein is catabolized as a metabolic fuel. The aim of the current study was to assess whether biochemical indices of protein synthesis and proteolytic mechanisms were altered in cuttlefish, Sepia officinalis, starved for 7 days. In mantle muscle, food deprivation is associated with a decrease in protein synthesis, as indicated by a decrease in the total RNA level and dephosphorylation of key signaling molecules, such as the eukaryote binding protein, 4E-BP1 (regulator of translation) and Akt. The ubiquitination-proteasome system (UPS) is activated as shown by an increase in the levels of proteasome β-subunit mRNA, polyubiquitinated protein, and polyubiquitin mRNA. As well, cathepsin activity levels are increased, suggesting increased proteolysis through the lysosomal pathway. Together, these mechanisms could supply amino acids as metabolic fuels. In gill, the situation is quite different. It appears that during the first stages of starvation, both protein synthesis and protein degradation are enhanced in gill. This is based upon increased phosphorylation of 4E-BP1 and enhanced levels of UPS indicators, especially 20S proteasome activity and polyubiquitin mRNA. It is proposed that an increased protein turnover is related to gill remodeling perhaps to retain essential hemolymph-borne compounds.     

Current concepts: III. Molecular aspects of dietary modulation of transcription and enhanced longevity

Dietary restriction is a known means of prolonging the life span of animals. How diet can increase longevity at the molecular level is not yet known. As organisms age, there is a decrease in the ability ot synthesize RNA and a decrease in protein synthesis indicating that there is an overall loss in gene expression. In addition, a decrease in protein turnover is evident indicating a lack of cellular renewal because of the accumulation of tissue protein. Evidence is presented in this review, that certain dietary regimens appear to be capable of enhancing the synthesis of mRNA and probably also produce enhanced turnover of tissue proteins. It is proposed that the physiological "stress" produced by restricted feeding paradigms can enhance gene expression and that this may be a significant factor in the maintenance of cellular homeostasis for a longer period of time.     

Autophagy, proteasomes, lipofuscin, and oxidative stress in the aging brain

In order to successfully respond to stress all cells rely on the ability of the proteasomal and lysosomal proteolytic pathways to continually maintain protein turnover. Increasing evidence suggests that as part of normal aging there are age-related impairments in protein turnover by the proteasomal proteolytic pathway, and perturbations of the lysosomal proteolytic pathway. Furthermore, with numerous studies suggest an elevated level of a specialized form of lysosomal proteolysis (autophagy or macroautophagy) occurs during the aging of multiple cell types. Age-related alterations in proteolysis are believed to contribute to a wide variety of neuropathological manifestations including elevations in protein oxidation, protein aggregation, and cytotoxicity. Within the brain altered protein turnover is believed to contribute to elevations in multiple forms of protein aggregation ranging from tangle and Lewy body formation, to lipofuscin-ceroid accumulation. In this review we discuss and summarize evidence for proteolytic alterations occurring in the aging brain, the contribution of oxidative stress to disruption of protein turnover during normal aging, the evidence for cross-talk between the proteasome and lysosomal proteolytic pathways in the brain, and explore the contribution of altered proteolysis as a mediator of oxidative stress, neuropathology, and neurotoxicity in the aging brain.     

Antidepressant treatment effects on hippocampal protein turnover: Molecular and spatial insights from mass spectrometry

A major challenge in managing depression is that antidepressant drugs take a long time to exert their therapeutic effects. For the development of faster-acting treatments, it is crucial to get an improved understanding of the molecular mechanisms underlying antidepressant mode of action. Here, we used a novel mass spectrometry-based workflow to investigate how antidepressant treatment affects brain protein turnover at single-cell and subcellular resolution. We combined stable isotope metabolic labeling, quantitative Tandem Mass Spectrometry (qTMS) and Multi-isotope Imaging Mass Spectrometry (MIMS) to simultaneously quantify and image protein synthesis and turnover in hippocampi of mice treated with the antidepressant paroxetine. We identified changes in turnover of individual hippocampal proteins that reveal altered metabolism-mitochondrial processes and report on subregion-specific turnover patterns upon paroxetine treatment. This workflow can be used to investigate brain protein turnover changes in vivo upon pharmacological interventions at a resolution and precision that has not been possible with other methods to date. Our results reveal acute paroxetine effects on brain protein turnover and shed light on antidepressant mode of action.     

Efficiency of energy retention in genetically obese animals and in dietary-induced thermogenesis

Genetically obese rodents (ob/ob mice and fa/fa rats) and animals with dietary-induced thermogenesis represent two extremes in efficiency of energy retention: the former deposit dietary energy with high efficiency, whereas the later deposit dietary energy with low efficiency. These differences in efficiency of energy retention must, at the cellular level, be associated with changes in efficiency and/or rate of formation and/or utilization of ATP (and other high energy intermediates). Brown adipose tissue possesses a unique proton-conductance pathway that reduces the efficiency of ATP synthesis. It has been speculated that this pathway is suppressed in obese (ob/ob) mice and accelerated in rats with dietary-induced thermogenesis. Metabolic reactions that alter the rate of ATP utilization in animals include Na+, K+-ATPase and protein turnover. The concentration of Na+, K+-ATPase enzyme unites in skeletal muscle and liver of young adult obese (ob/ob) mice is lower than in tissues of young adult lean mice. There also appear to be alterations in protein turnover in certain tissues of obese (ob/ob) mice, but additional studies are required to determine if whole-body protein turnover is altered in these animals. Data are unavailable on either Na+, K+-ATPase or protein turnover in tissue of animals with dietary-induced thermogenesis. Continuation of studies in these areas should provide a metabolic basis for understanding individual variability in efficiency of energy retention.     

Membrane protein Crry maintains homeostasis of the complement system

Complement activation is tightly regulated to avoid excessive inflammatory and immune responses. Crry(-/-) is an embryonic lethal phenotype secondary to the maternal complement alternative pathway (AP) attacking a placenta deficient in this inhibitor. In this study, we demonstrate that Crry(-/-) mice could be rescued on a partial as well as on a complete factor B (fB)- or C3-deficient maternal background. The C3 and fB protein concentrations in Crry(-/-)C3(+/-) and Crry(-/-)fB(+/-) mice were substantially reduced for gene dosage secondary to enhanced AP turnover. Based on these observations, a breeding strategy featuring reduced maternal AP-activating capacity rescued the lethal phenotype. It led to a novel, stable line of Crry SKO mice carrying normal alleles for C3 and fB. Crry SKO mice also had accelerated C3 and fB turnover and therefore reduced AP- activating potential. These instructive results represent an example of a membrane regulatory protein being responsible for homeostasis of the complement system. They imply that there is constant turnover on cells of the AP pathway which functions as an immune surveillance system for pathogens and altered self.     

Hydrocortisone regulates arylsulfatase A (cerebroside-3-sulfate-3-sulfohydrolase) by decreasing the quantity of the enzyme in cultures of cells dissociated from embryonic mouse cerebra

Previous work from our laboratory (Biochem. J. 219:689–697 (1984) had shown that hydrocortisone stimulated the net accumulation of the myelin-specific sulfolipid in cultures of cells dissociated from embryonic mouse cerebra. This accumulation caused by hydrocortisone was shown to be due to a decrease of sulfolipid degradation by arylsulfatase A (ASA) and not due to a stimulation of its synthesis by a sulfotransferase. Both ASA activity and the turnover of sulfolipid were decreased by hydrocortisone to 60–62% of untreated cells. In current work the same decrease in enzyme activity was obtained and enzyme linked immunosorbent assays demonstrate that hydrocortisone decreased the number of ASA protein molecules to 61% of untreated cells [(-)hydrocorcortisone 0.31±0.06 ng ASA/g protein; (+)hydrocortisone: 0.18±0.04 ng ASA/g protein]. This decrease in the number of ASA molecules correlates well with the decrease in both the enzyme activity and the sulfolipid turnover, which suggests that the major mode of inhibition of ASA activity by hydrocortisone involves a decrease in the concentration of ASA in the cells rather than some other mechanism of inhibition.The material in this paper has been included in a dissertation submitted by A.J.M. in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Temple University.     

A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradation

Background

Alterations in multiple cellular pathways contribute to the development of chronic neurodegeneration such as a sporadic Alzheimer's disease (AD). These, in turn, involve changes in gene expression, amongst which are genes regulating protein processing and turnover such as the components of the ubiquitin-proteosome system. Recently, we have identified a cDNA whose expression was altered in AD brains. It contained an open reading frame of 247 amino acids and represented a novel RING finger protein, RNF182. Here we examined its biochemical properties and putative role in brain cells.

Results

RNF182 is a low abundance cytoplasmic protein expressed preferentially in the brain. Its expression was elevated in post-mortem AD brain tissue and the gene could be up regulated in vitro in cultured neurons subjected to cell death-inducing injuries. Subsequently, we have established that RNF182 protein possessed an E3 ubiquitin ligase activity and stimulated the E2-dependent polyubiquitination in vitro. Yeast two-hybrid screening, overexpression and co-precipitation approaches revealed, both in vitro and in vivo, an interaction between RNF182 and ATP6V0C, known for its role in the formation of gap junction complexes and neurotransmitter release channels. The data indicated that RNF182 targeted ATP6V0C for degradation by the ubiquitin-proteosome pathway. Overexpression of RNF182 reduced cell viability and it would appear that by itself the gene can disrupt cellular homeostasis.

Conclusion

Taken together, we have identified a novel brain-enriched RING finger E3 ligase, which was up regulated in AD brains and neuronal cells exposed to injurious insults. It interacted with ATP6V0C protein suggesting that it may play a very specific role in controlling the turnover of an essential component of neurotransmitter release machinery.     

Enzymatic properties of the RecA803 protein, a partial suppressor of recF mutations.

The RecA803 protein suppresses the recombinational repair defect of recF mutations and displays enhanced joint molecule formation in vitro (Madiraju et al., 1988). To understand the physical basis for these phenomena, the biochemical properties of RecA803 protein were compared with those of the wild-type protein. The RecA803 protein shows greater DNA-dependent ATPase activity than the wild-type protein with either M13 single-stranded (ss) DNA, which contains secondary structure, or double-stranded DNA. This increased activity reflects an enhanced ability of the mutant protein to form active complexes with these DNA molecules rather than an enhanced catalytic turnover activity, because identical kcat values for ATP hydrolysis are obtained when DNA substrates lacking secondary structure are examined. In addition, the ssDNA-dependent ATPase activity of RecA803 protein displays greater resistance to inhibition by SSB (single-stranded DNA binding) protein. These properties of the RecA803 protein are not due to either an increased binding affinity for ssDNA or an increased kinetic lifetime of RecA803 protein-ssDNA complexes, demonstrating that altered protein-DNA stability is not the basis for the enhanced properties of RecA803 protein. However, the nucleation-limited rate of association with ssDNA is more rapid for the RecA803 protein than for wild-type RecA protein. Consequently, we suggest that altered protein-protein interactions may account for the differences between these two proteins. The implications of these results with regard to the partial suppression of recF mutations by recA803 are discussed (Madiraju et al., 1988).     

Microhydrodynamics simulation of protein crystallization. I. Static calculations.

A computer simulation method is proposed to study the effects of hydrodynamic interactions on protein crystallization. It is a combination of Stokesian dynamics and continuum hydrodynamics and is referred to as "microhydrodynamics." The method is checked against analytical expressions for Stokes drag and diffusion coefficients for unit spheres. For a number of protein molecules the diffusion coefficients have been calculated and compared with experimental values. It is shown that the method works well for stationary calculations. Using dynamical calculations interacting protein molecules will be simulated to study the events in the early stages of protein crystallization.     

Restriction of energy intake, energy expenditure, and aging

Energy restriction (ER), without malnutrition, increases maximum life span and retards the development of a broad array of pathophysiological changes in laboratory rodents. The mechanism responsible for the retardation of aging by ER is, however, unknown. One proposed explanation is a reduction in energy expenditure (EE). Reduced EE may increase life span by decreasing the number of oxygen molecules interacting with mitochondria, thereby lowering reactive oxygen species (ROS) production. As a step toward testing this hypothesis, it is important to determine the effect of ER on EE. Several whole-body, organ, and cellular studies have measured the influence of ER on EE. In general, whole-body studies have reported an acute decrease in mass-adjusted EE that disappears with long-term ER. Organ-specific studies have shown that decreases in EE of liver and gastrointestinal tract are primarily responsible for initial reductions in EE with ER. These data, however, do not determine whether cellular EE is altered with ER. Three major processes contributing to resting EE at the cellular level are mitochondrial proton leak, Na(+)-K(+)-ATPase activity, and protein turnover. Studies suggest that proton leak and Na(+)-K(+)-ATPase activity are decreased with ER, whereas protein turnover is either unchanged or slightly increased with ER. Thus, two of the three major processes contributing to resting EE at the cellular level may be decreased with ER. Although additional cellular measurements are needed, the current results suggest that a lowering of EE could be a mechanism for the action of ER.