Sharon W. Lin

Degradation of oxidatively denatured proteins in Escherichia coli.

When exposed to oxidative stress, by oxygen radicals or H2O2, E. coli exhibited decreased growth, decreased protein synthesis, and dose-dependent increases in protein degradation. The quinone menadione induced proteolysis when cells were incubated in air, but was not effective when cells were incubated without oxygen. Anaerobically grown cells also exhibited significantly lower proteolytic capacity than did cells that were grown aerobically. Xanthine plus xanthine oxidase (which generate O2- and H2O2) caused a stimulation of proteolysis which was inhibitable by catalase, but not by superoxide dismutase: Indicating that H2O2 was responsible for the increased protein degradation. Indeed, H2O2 alone was effective in inducing increased intracellular proteolysis. Two-dimensional polyacrylamide gel electrophoresis of [3H]leucine labeled E. coli revealed greater than 50% decreases in the concentrations of 10-15 cell proteins following H2O2 or menadione exposure, while several other proteins were less severely affected. To test for the presence of soluble proteases, we prepared cell-free extracts of E. coli and incubated them with radio-labeled protein substrates. E. coli extracts degraded casein and globin polypeptides at rapid rates but showed little activity with native proteins such as superoxide dismutase, hemoglobin, bovine serum albumin, or catalase. When these same proteins were denatured by exposure to oxygen radicals or H2O2, however, they became excellent substrates for degradation in E. coli extracts. Studies with albumin revealed correlations greater than 0.95 between the degree of oxidative denaturation and proteolytic susceptibility. Pretreatment of E. coli with menadione or H2O2 did not increase the proteolytic capacity of cell extracts; indicating that neither protease activation, nor protease induction were required.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteasome Inhibition and Aggresome Formation in Sporadic Inclusion-Body Myositis and in Amyloid-β Precursor Protein-Overexpressing Cultured Human Muscle Fibers

The 26S proteasome system is involved in eliminating various proteins, including ubiquitinated misfolded/unfolded proteins, and its inhibition results in cellular accumulation of protein aggregates. Intramuscle-fiber ubiquitinated multiprotein-aggregates are characteristic of sporadic inclusion-body myositis (s-IBM) muscle fibers. Two major types of aggregates exist, containing either amyloid-beta (Abeta) or phosphorylated tau (p-tau). We have now asked whether abnormalities of the 26S proteasome contribute to s-IBM pathogenesis and whether the multiprotein aggregates have features of aggresomes. Using cultured human muscle fibers we also studied the effect of amyloid-beta precursor protein (AbetaPP) overexpression on proteasome function and the influence of proteasome inhibition on aggresome formation. We report that in s-IBM muscle biopsies 26S proteasome subunits were immunodetected in the gamma-tubulin-associated aggresomes, which also contained Abeta, p-tau, ubiquitin, and HSP70. In addition, a) expression of proteasome subunits was greatly increased, b) the 20Salpha proteasome subunit co-immunoprecipitated with AbetaPP/Abeta, and c) the three major proteasomal proteolytic activities were reduced. In cultured muscle fibers, AbetaPP-overexpressing fibers displayed diminished proteasomal proteolytic activities, and addition of proteasome inhibitor strikingly increased aggresome formation. Accordingly, proteasome dysfunction in s-IBM muscle fibers may play a role in accumulation of misfolded, potentially cytotoxic proteins and may be induced by increased intracellular AbetaPP/Abeta.

Superoxide dismutase is preferentially degraded by a proteolytic system from red blood cells following oxidative modification by hydrogen peroxide.

The cupro-zinc enzyme superoxide dismutase (SOD) undergoes an irreversible (oxidative) inactivation when exposed to its product, hydrogen peroxide (H2O2). Recent studies have shown that several oxidatively modified proteins (e.g., hemoglobin, albumin, catalase, etc.) are preferentially degraded by a novel proteolytic pathway in the red blood cell. We report that bovine SOD is oxidatively inactivated by exposure to H2O2, and that the inactivated enzyme is selectively degraded by proteolytic enzymes in cell-free extracts of bovine erythrocytes. For example, 95% inactivation of SOD by 1.5 mM H2O2 was accompanied by a 106 fold increase in the proteolytic susceptibility of the enzyme during (a subsequent) incubation with red cell extract. Both SOD inactivation and proteolytic susceptibility increased with H2O2 concentration and/or time of exposure to H2O2. Pre-incubation of red cell extracts with metal chelators, serine reagents, or sulfhydryl reagents inhibited the (subsequent) preferential degradation of H2O2-modified SOD. Furthermore, a slight inhibition of degradation was observed with the addition of ATP. We suggest that H2O2-inactivated SOD is recognized and preferentially degraded by the same. ATP-independent, metallo- serine- and sulfhydryl- proteinase pathway which degrades other oxidatively denatured red cell proteins. Related work in this laboratory suggests that this novel proteolytic pathway may actually consist of a 700 kDa enzyme complex of proteolytic activities. Mature red cells have no capacity for de novo protein synthesis but do have extremely high concentrations of SOD. Red cell SOD generates (and is, therefore, exposed to) H2O2 on a continuous basis, by dismutation of superoxide (from hemoglobin autooxidation and the interaction of hemoglobin with numerous xenobiotics).(ABSTRACT TRUNCATED AT 250 WORDS)

Lens proteasome shows enhanced rates of degradation of hydroxyl radical modified alpha-crystallin.

Proteasome, a high molecular weight protease complex (HMP, approximately 600 kDa) was isolated from bovine eye lens epithelium tissue. In contrast with prior reports, lens proteasome degraded the major lens protein alpha-crystallin and S-carboxymethylated bovine serum albumin at 37 degrees C, mostly to trichloroacetic acid precipitable polypeptides. The proteasome, thus isolated, was labile at 55 degrees C. As indicated by the ability of p-chloromercuribenzoate and N-ethylmaleimide to block activity, a thiol group is required for activity. Alpha-crystallin was oxidized by exposure to 60Co-irradiation under an atmosphere of N2O (1-50 kilorads). This dose delivered 0.1-5.7 mol of hydroxyl radicals per mol of crystallin. Irradiation resulted in increased heterogeneity, aggregation, and fragmentation of the crystallin preparation. The proteolytic susceptibility of alpha-crystallin to the lens HMP was enhanced by the irradiation in a dose-dependent manner up to 20 kilorads (.OH concentration up to 2.3 mol per mol of alpha-crystallin). When 50 kilorads (5.7 mol .OH per mol of alpha-crystallin) was used, there was extensive aggregation and no enhancement in proteolysis over the unirradiated sample. The data indicate that the lens HMP can degrade mildly photooxidized lens proteins, but proteins which are extensively damaged are not degraded and may accumulate. This may be related to cataract formation.

Iron Causes Interactions of TAK1, p21ras, and Phosphatidylinositol 3-Kinase in Caveolae to Activate IκB Kinase in Hepatic Macrophages

We recently discovered a novel signaling phenomenon involving a rapid and transient rise in intracellular low molecular weight iron complex(es) in activation of IκB kinase (IKK) in hepatic macrophages. We also showed direct treatment with ferrous iron substitutes for this event to activate IKK. The present study used this model to identify upstream kinases responsible for IKK activation. IKK activation induced by iron is abrogated by overexpression of a dominant negative mutant (DN) for transforming growth factor β-activated kinase-1 (TAK1), NF-κB-inducing kinase, or phosphatidylinositol 3-kinase (PI3K) and by treatment with the mitogen-activated protein kinase (MAPK) kinase-1 (MEK1) inhibitor. Iron increases AKT phosphorylation that is prevented by DNTAK1 or DNp21ras. Iron causes ERK1/2 phosphorylation that is attenuated by DN-PI3K, prevented by DNp21ras, but unaffected by DNTAK1. Iron-induced TAK1 activity is not affected by the PI3K or MEK1 inhibitor, suggesting TAK1 is upstream of PI3K and MEK1. Iron increases interactions of TAK1 and PI3K with p21ras as demonstrated by co-immunoprecipitation and co-localization of these proteins with caveolin-1 as shown by immunofluorescent microscopy. Finally, filipin III, a caveolae inhibitor, abrogates iron-induced TAK1 and IKK activation. In conclusion, MEK1, TAK1, NF-κ-inducing kinase, and PI3K are required for iron-induced IKK activation in hepatic macrophages and TAK1, PI3K, and p21ras physically interact in caveolae to initiate signal transduction.

Continuous versus Cyclic Progesterone Exposure Differentially Regulates Hippocampal Gene Expression and Functional Profiles

This study investigated the impact of chronic exposure to continuous (CoP4) versus cyclic progesterone (CyP4) alone or in combination with 17β-estradiol (E2) on gene expression profiles targeting bioenergetics, metabolism and inflammation in the adult female rat hippocampus. High-throughput qRT-PCR analyses revealed that ovarian hormonal depletion induced by ovariectomy (OVX) led to multiple significant gene expression alterations, which were to a great extent reversed by co-administration of E2 and CyP4. In contrast, co-administration of E2 and CoP4 induced a pattern highly resembling OVX. Bioinformatics analyses further revealed clear disparities in functional profiles associated with E2+CoP4 and E2+CyP4. Genes involved in mitochondrial energy (ATP synthase α subunit; Atp5a1), redox homeostasis (peroxiredoxin 5; Prdx5), insulin signaling (insulin-like growth factor I; Igf1), and cholesterol trafficking (liver X receptor α subtype; Nr1h3), differed in direction of regulation by E2+CoP4 (down-regulation relative to OVX) and E2+CyP4 (up-regulation relative to OVX). In contrast, genes involved in amyloid metabolism (β-secretase; Bace1) differed only in degree of regulation, as both E2+CoP4 and E2+CyP4 induced down-regulation at different efficacy. E2+CyP4-induced changes could be associated with regulation of progesterone receptor membrane component 1(Pgrmc1). In summary, results from this study provide evidence at the molecular level that differing regimens of hormone therapy (HT) can induce disparate gene expression profiles in brain. From a translational perspective, confirmation of these results in a model of natural menopause, would imply that the common regimen of continuous combined HT may have adverse consequences whereas a cyclic combined regimen, which is more physiological, could be an effective strategy to maintain neurological health and function throughout menopausal aging.

17β-estradiol and progesterone regulate expression of β-amyloid clearance factors in primary neuron cultures and female rat brain.

The accumulation of β-amyloid protein (Aβ) is a key risk factor in the development of Alzheimers disease. The ovarian sex steroid hormones 17β-estradiol (E(2)) and progesterone (P(4)) have been shown to regulate Aβ accumulation, although the underlying mechanism(s) remain to be fully elucidated. In this study, we investigate the effects of E(2) and P(4) treatment on the expression levels of Aβ clearance factors including insulin-degrading enzyme, neprilysin, endothelin-converting enzyme 1 and 2, angiotensin-converting enzyme, and transthyretin, both in primary neuron cultures and female rat brains. Our results show that E(2) and P(4) affect the expression levels of several Aβ clearance factors in dose- and time-dependent manners. Most notably, expression of insulin-degrading enzyme is significantly increased by both hormones in cultured neurons and in vivo and is inversely associated with the soluble Aβ levels in vivo. These findings further define sex steroid hormone actions involved in regulation of Aβ, a relationship potentially important to therapeutic approaches aimed at reducing risk of Alzheimers disease.

Differential Responses of Progesterone Receptor Membrane Component-1 (Pgrmc1) and the Classical Progesterone Receptor (Pgr) to 17β-Estradiol and Progesterone in Hippocampal Subregions that Support Synaptic Remodeling and Neurogenesis

Progesterone (P4) and estradiol (E2) modulate neurogenesis and synaptic remodeling in the hippocampus during the rat estrous cycle and in response to deafferenting lesions, but little is known about the steroidal regulation of hippocampal progesterone receptors associated with these processes. We examined the neuronal expression of progesterone receptor membrane component-1 (Pgrmc1) and the classical progesterone receptor (Pgr), by in situ hybridization and immunohistochemistry. Pgr, a transcription factor, has been associated with synaptic remodeling and other major actions of P4, whereas Pgrmc1 is implicated in P4-dependent proliferation of adult neuroprogenitor cells and with rapid P4 effects on membranes. Ovariectomized adult rats were given E2, P4, or E2+P4 on two schedules: a 4-d model of the rodent estrous cycle and a 30-d model of postmenopausal hormone therapy. Pgr was hormonally responsive only in CA1 pyramidal neurons, and the induction of Pgr by E2 was partly antagonized by P4 only on the 30-d schedule. In CA3 pyramidal and dentate gyrus (DG) neurons, Pgr was largely unresponsive to all hormone treatments. In contrast to Pgr, Pgrmc1 was generally induced by E2 and/or P4 throughout the hippocampus in CA1, CA3, and DG neurons. In neuroprogenitor cells of the DG (immunopositive for bromodeoxyuridine and doublecortin), both Pgrmc1 and Pgr were detected. The differential regulation of hippocampal Pgrmc1 and Pgr by E2 and P4 may guide drug development in hormonal therapy for support of neurogenesis and synaptic regeneration.

Age Increase of Estrogen Receptor-α (ERα) in Cortical Astrocytes Impairs Neurotrophic Support in Male and Female Rats

Rodent models show decreased neuronal responses to estradiol (E2) during aging (E2-desensitization) in association with reduced neuronal estrogen receptor (ER)-α, but little is known about age changes of E2-dependent astrocytic neurotrophic support. Because elevated expression of astrocyte glial fibrillary acidic protein (GFAP) is associated with impaired neurotrophic activity and because the GFAP promoter responds to ERα, we investigated the role of astrocytic ERα and ERβ in impaired astrocyte neurotrophic activity during aging. In vivo and in vitro, ERα was increased greater than 50% with age in astrocytes from the cerebral cortex of male rats (24 vs 3 months), whereas ERβ did not change. In astrocytes from 3-month-old males, experimentally increasing the ERα to ERβ ratio induced the aging phenotype of elevated GFAP and impaired E2-dependent neurite outgrowth. In 24-month-old male astrocytes, lowering ERα reversed the age elevation of GFAP and partially restored E2-dependent neurite outgrowth. Mixed glia (astrocytes to microglia, 3:1) of both sexes also showed these age changes. In a model of perimenopause, mixed glia from 9- to 15-month rats showed E2 desensitization: 9-month regular cyclers retained young-like ERα to ERβ ratios and neurotrophic activity, whereas 9-month noncyclers had elevated ERα and GFAP but low E2-dependent neurotrophic activity. In vivo, ERα levels in cortical astrocytes were also elevated. The persisting effects of ovarian acyclicity in vitro are hypothesized to arise from steroidal perturbations during ovarian senescence. These findings suggest that increased astrocyte ERα expression during aging contributes to the E2 desensitization of the neuronal responses in both sexes.