Inhibition of rabbit reticulocyte lysate protein synthesis by heavy metal ions involves the phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2

The effect of heavy metal ions (in particular Cd2+, Hg2+, and Pb2+) on protein synthesis in hemin-supplemented reticulocyte lysates was investigated. Heavy metal ions were found to inhibit protein synthesis in hemin-supplemented lysates with biphasic kinetics. The shut off of protein synthesis occurred in conjunction with the phosphorylation of the alpha-subunit of the eukaryotic initiation factor (eIF) 2, the loss of reversing factor (RF) activity, and the disaggregation of polyribosomes. Addition of eIF-2 or RF to heavy metal ion-inhibited lysates restored protein synthesis to levels observed in hemin-supplemented controls. The stimulation of protein synthesis observed upon the addition of cAMP to heavy metal ion-inhibited lysates correlated with the inhibition of eIF-2 alpha phosphorylation and the restoration of RF activity. The partial restoration of protein synthesis observed upon the addition of MgGTP to heavy metal ion-inhibited lysates correlated with a partial inhibition of eIF-2 alpha phosphorylation. Addition of glucose 6-phosphate was found to have no effect on protein synthesis of eIF-2 alpha phosphorylation under these conditions. Antiserum raised to the reticulocyte heme-regulated eIF-2 alpha kinase inhibited the phosphorylation of eIF-2 alpha catalyzed by Hg2+-inhibited lysate. The inhibition of protein synthesis observed in the presence of heavy metal ions correlated with the relative biological toxicity of the ions. Highly toxic ions (AsO-2, Cd2+, Hg2+, Pb2+) inhibited protein synthesis by 50% at concentrations of 2.5-10 microM. Cu2+, Fe3+, and Zn2+, which are moderately to slightly toxic ions, inhibited protein synthesis by 50% at concentrations of 40, 250, and 300 microM, respectively. The data presented here indicate that heavy metal ions inhibit protein chain initiation in hemin-supplemented lysates by stimulating the phosphorylation of eIF-2 alpha apparently through the activation of the heme-regulated eIF-2 alpha kinase rather than through inhibition of the rate of eIF-2 alpha dephosphorylation.     

Chest-wall deformity after tissue expansion for breast reconstruction

A prospective longitudinal study of chest-wall deformity after tissue expansion for breast reconstruction was performed in 19 women. CT imaging was a sensitive method for detecting occult deformity. Using a semiquantitative scale for measuring deformity, all patients and 94 percent of expanders had some thoracic abnormality after tissue expansion. Rib and chest-wall contour changes were observed under 81 and 68 percent of the expanders, respectively. Routine chest roentgenograms were not a sensitive method for evaluating these deformities. The magnitude of deformity after unilateral expansion was not significantly different from that after bilateral expansion. Linear regression analysis indicated that early periprosthetic capsular contracture was negatively correlated with chest wall deformity. Only one patient experienced a clinically noticeable complication from chest compression--transient postexpansion exertional dyspnea. After removing the expanders and placing permanent implants along with capsulotomy, the mean deformity index decreased by 57 percent after 10.5 months median follow-up, which was highly significant (p less than 0.001). Our findings suggest that chest-wall deformity is a common occurrence after tissue expansion in patients undergoing breast reconstruction and is usually of minor clinical significance.     

The outcome of poliovirus infections in K562 cells is cytolytic rather than persistent after hemin-induced differentiation.

K562-Mu erythroleukemia cells readily establish a long-term persistent poliovirus infection characterized by continuous virus production in the absence of complete p220 cleavage and host translation shutoff (R. E. Lloyd and M. Bovee, Virology 194:200-209, 1993). The mechanism of resistance appears to be modulated at the intracellular level and to be related to decreased virus-mediated cytopathic effects (P. A. Benton, J. W. Murphy, and R. E. Lloyd Virology 213:7-18, 1995). It is well documented that hemin induces the differentiation of K562 cells and alters the expression of several host proteins. We report here that growth of K562 cells in hemin prior to poliovirus infection results in a dose-dependent increase in virus-induced cell lysis and thereby alters the normally persistent outcome of infection to a more lytic phenotype. K562 cells infected after hemin treatment displayed increased host translation shutoff, p220 cleavage, viral protein synthesis, and viral RNA accumulation compared with nontreated cells. Since hemin treatment of K562 cells also induced the increased expression of several heat shock proteins (Hsp70, Hsc70, Hsp90, and cohort p60), we tested the hypothesis that their increased expression may play a role in altering poliovirus infection in hemin-treated K562 cells. However, neither heat stress nor oxidative stress, inducers of heat shock protein synthesis, altered the outcome (of virus infections. In addition, we report the novel finding that subunits of two translation initiation factors, p220 (eIF-4G) and eIF-2alpha, are cleaved as a result of hemin treatment of K562 cells. It is proposed that hemin alters the expression of specific host proteins in K562 cells, probably other than heat shock proteins, which changes the initial response to poliovirus infections from persistent to lytic.     

Toxic heavy metal ions activate the heme-regulated eukaryotic initiation factor-2 alpha kinase by inhibiting the capacity of hemin-supplemented reticulocyte lysates to reduce disulfide bonds.

Addition of toxic heavy metal ions (Cd2+, Hg2+, and Pb2+) to hemin-supplemented rabbit reticulocyte lysate brings about the activation of the heme-regulated eukaryotic initiation factor 2 alpha kinase (HRI) and the inhibition of protein chain initiation. In this report we examined the effects of monothiol and dithiol compounds, metal ion-chelating agents, and metallothioneins (MT) on metal ion-induced inhibition of protein synthesis. The dithiol compounds dithiothreitol and 2,3-dimercaptopropane sulfonic acid prevented and relieved the inhibition of protein synthesis caused by Cd2+ and Hg2+ in hemin-supplemented lysates, but the monothiol compounds 2-mercaptoethanol, cysteamine, D-(-)penicillamine, and glutathione had no effect. The inhibition of protein synthesis caused by Cd2+ was reversed by the addition of excess EDTA but not by the addition of excess nitrilotriacetic acid. Toxic heavy metal ions inhibited the capacity of hemin-supplemented lysate to reduce disulfide bonds. Addition of excess EDTA to Cd(2+)-inhibited lysates restored the capacity of the lysate to reduce disulfide bonds and inhibited the phosphorylation of eukaryotic initiation factor eIF-2. MTs and their apoproteins (apoMTs) inhibited the activation of HRI and protected protein synthesis from inhibition by Cd2+, Hg2+, and Pb2+. Addition of apoMTs to heavy metal ion-inhibited lysates restored the capacity of lysates to reduce disulfide bonds. The restoration of the lysate's thioredoxin/thioredoxin reductase activity was accompanied by the inactivation of HRI and the resumption of protein synthesis, indicating that apoMTs can "detoxify" metal ions already bound to proteins. Several observations presented in this report suggest that the binding of metal ions to the alpha-domain of MT is responsible for the ability of MT to sequester bound metal in a non-toxic form. Addition of glucose 6-phosphate or NADPH had no effect on protein synthesis in metal ion-inhibited lysates, and NADPH concentrations in Cd(2+)-inhibited and hemin-supplemented control lysates were equivalent. The data suggest that the metal ions cause the inhibition of protein synthesis by binding to vicinal sulfhydryl groups present in some critical protein(s), possibly the dithiols present in the active site of thioredoxin and (or) thioredoxin reductase, which leads to the activation of HRI.     

Molybdate inhibits hsp90, induces structural changes in its C-terminal domain, and alters its interactions with substrates

To examine the biochemical mechanism by which hsp90 exerts its essential positive function on certain signal transduction proteins, we characterized the effects of molybdate and geldanamycin on hsp90 function and structure. Molybdate inhibited hsp90-mediated p56lck biogenesis and luciferase renaturation while enforcing salt-stable interactions with these substrates. Molybdate also reduced the amount of free hsp90 present in cell lysates, inhibited hsp90's ability to bind geldanamycin, and induced resistance to proteolysis at a specific region within the C-terminal domain of hsp90. In contrast, the hsp90 inhibitor geldanamycin prevented hsp90 from assuming natural or molybdate-induced conformations that allow salt-stable interactions with substrates. When these compounds were applied sequentially, the order of addition determined the effects observed, indicating that these agents had opposing effects on hsp90. We conclude that a specific region within the C-terminal domain of hsp90 (near residue 600) determines the mode by which hsp90 interacts with substrates and that the ability of hsp90 to cycle between alternative modes of interaction is obligatory for hsp90 function.     

A calorimetric study of binary mixtures of dihydrosphingomyelin and sterols,sphingomyelin, or phosphatidylcholine

The thermotropic properties of binary mixtures of D-erythro-n-palmitoyl-dihydrosphingomyelin (16:0-DHSM), D-erythro-n-palmitoyl-sphingomyelin (16:0-SM), cholesterol, lathosterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were studied by differential scanning calorimetry. Addition of sterol to 16:0-DHSM and 16:0-SM bilayers resulted in a progressive decrease in both the T(m) and the enthalpy of the main transition. The sterol-induced broad components in 16:0-DHSM endotherms had markedly lower enthalpies than those induced in 16:0-SM. Pretransitions recorded in 16:0-DHSM and 16:0-SM membranes responded differently to low concentrations of cholesterol. The presence of 5 mol % cholesterol increased the pretransition temperature in 16:0-SM bilayers, whereas it decreased the temperature in 16:0-DHSM membranes. Lathosterol behaved in general as cholesterol with regard to its effects on the thermotropic behavior of both sphingolipids, but it appeared to form more stable sterol-rich domains, as seen from the higher T(m) of the broad component, in comparison to cholesterol. Thermograms recorded on binary mixtures of 16:0-SM:16:0-DHSM and DPPC:16:0-DHSM showed that 16:0-SM mixed nearly ideally with 16:0-DHSM, whereas DPPC mixing was less ideal in a 16:0-DHSM membrane. In conclusion, we observed that 16:0-DHSM interactions with sterols differed from that seen with 16:0-SM, and that 16:0-DHSM mixed better with 16:0-SM than DPPC, which indicates that DHSM could function as a membrane organizer within laterally condensed domains.     

Differential inhibition of Hsc70 activities by two Hsc70-binding peptides

The ability of two high-affinity Hsc70-binding peptides [FYQLALT (peptide-Phi) and NIVRKKK (peptide-K)] to differentially inhibit Hsc70-dependent processes in rabbit reticulocyte lysate (RRL) was examined. Both peptide-Phi and peptide-K inhibited chaperone-dependent renaturation of luciferase in RRL. Peptide-Phi, but not peptide-K, blocked Hsp90/Hsc70-dependent transformation of the heme-regulated eIF2 alpha kinase (HRI) into an active, heme-regulatable kinase. In contrast, peptide-K, but not peptide-Phi, inhibited Hsc70-mediated suppression of the activation of mature-transformed HRI. Furthermore, HDJ2 (Human DnaJ homologue 2), but not HDJ1, potentiated the ability of Hsc70 to suppress the activation of HRI in RRL. Mechanistically, peptide-K inhibited, while peptide-Phi enhanced, HDJ2-induced stimulation of Hsc70 ATPase activity in vitro. The data presented support the hypotheses that peptide-Phi acts to inhibit Hsc70 function by binding to the hydrophobic peptide-binding cleft of Hsc70, while peptide-K acts through binding to a site that modulates the interaction of Hsc70 with DnaJ homologues. Overall, the data indicate that peptide-Phi and peptide-K have differential effects on Hsc70 functions under quasi-physiological conditions in RRL, and suggest that therapeutically valuable peptide mimetics can be designed to inhibit specific functions of Hsc70.     

Expression of Bcl-2, Bcl-x, Mcl-1, Bax and Bak in human uterine leiomyomas and myometrium during the menstrual cycle and after menopause

To investigate the expression of Bcl-2, Bcl-x, Mcl-1, Bax and Bak proteins in human uterine leiomyomas and homologous myometrium during the menstrual cycle and after menopause.The expression of Bcl-2, Bcl-x, Mcl-1, Bax and Bak in leiomyomas (n=24) and myometrial samples (n=22) from women with leiomyomas was measured by immunohistochemistry and Western blot. Measured by immunohistochemistry, a significant difference between leiomyomas and myometrium was observed only for the Bax protein, in tissues obtained from women in the secretory phase of the menstrual cycle. The Bcl-2 staining was more abundant in leiomyomas than in myometrium only in tissues obtained in the proliferative phase of the cycle. Bcl-2 was more abundant in leiomyomas from women of fertile age than in leiomyomas from menopausal women. No significant differences were observed for the Bcl-x or Bak proteins, whereas the Mcl-1 protein was significantly less abundant in secretory phase leiomyomas than in leiomyomas from menopausal women. Western blot analysis based on pools of tissue extracts from the different groups essentially confirmed the data obtained by immunohistochemistry. Bcl-2 family proteins are expressed in leiomyomas and myometrium in different phases related to and influenced by gonadal steroids. These proteins are suggested to interact with each other in the regulation of programmed cell death, apoptosis, but their specific role in growth control of uterine leiomyomas remains to be investigated.