Ronald Jemmerson

Induction of Apoptotic Program in Cell-Free Extracts: Requirement for dATP and Cytochrome c

A cell-free system based on cytosols of normally growing cells is established that reproduces aspects of the apoptotic program in vitro. The apoptotic program is initiated by addition of dATP. Fractionation of cytosol yielded a 15 kDa protein that is required for in vitro apoptosis. The absorption spectrum and protein sequence revealed that this protein is cytochrome c. Elimination of cytochrome c from cytosol by immunodepletion, or inclusion of sucrose to stabilize mitochondria during cytosol preparation, diminished the apoptotic activity. Adding back cytochrome c to the cytochrome c-depleted extracts restored their apoptotic activity. Cells undergoing apoptosis in vivo showed increased release of cytochrome c to their cytosol, suggesting that mitochondria may function in apoptosis by releasing cytochrome c.

Calcium-induced cytochrome c release from CNS mitochondria is associated with the permeability transition and rupture of the outer membrane.

The mechanisms of Ca2+‐induced release of Cytochrome c (Cyt c) from rat brain mitochondria were examined quantitatively using a capture ELISA. In 75 or 125u2003mm KCl‐based media 1.4u2003µmol Ca2+/mg protein caused depolarization and mitochondrial swelling. However, this resulted in partial Cyt c release only in 75u2003mm KCl. The release was inhibited by Ru360, an inhibitor of the Ca2+ uniporter, and by cyclosporin A plus ADP, a combination of mitochondrial permeability transition inhibitors. Transmission electron microscopy (TEM) revealed that Ca2+‐induced swelling caused rupture of the outer membrane only in 75u2003mm KCl. Koenigs polyanion, an inhibitor of mitochondrial porin (VDAC), enhanced swelling and amplified Cyt c release. Dextran T70 that is known to enhance mitochondrial contact site formation did not prevent Cyt c release. Exposure of cultured cortical neurons to 500u2003µm glutamate for 5u2003min caused Cyt c release into the cytosol 30u2003min after glutamate removal. MK‐801 or CsA inhibited this release. Thus, the release of Cyt c from CNS mitochondria induced by Ca2+in vitro as well as in situ involved the mPT and appeared to require the rupture of the outer membrane.

The Proapoptotic Function of Noxa in Human Leukemia Cells Is Regulated by the Kinase Cdk5 and by Glucose

The BH3-only protein, Noxa, is induced in response to apoptotic stimuli, such as DNA damage, hypoxia, and proteasome inhibition in most human cells. Noxa is constitutively expressed in proliferating cells of hematopoietic lineage and required for apoptosis in response to glucose stress. We show that Noxa is phosphorylated on a serine residue (S(13)) in the presence of glucose. Phosphorylation promotes its cytosolic sequestration and suppresses its apoptotic function. We identify Cdk5 as the Noxa kinase and show that Cdk5 knockdown or expression of a Noxa S(13) to A mutant increases sensitivity to glucose starvation, confirming that the phosphorylation is protective. Both glucose deprivation and Cdk5 inhibition promote apoptosis by dephosphorylating Noxa. Paradoxically, Noxa stimulates glucose consumption and may enhance glucose turnover via the pentose phosphate pathway rather than through glycolysis. We propose that Noxa plays both growth-promoting and proapoptotic roles in hematopoietic cancers with phospho-S(13) as the glucose-sensitive toggle switch controlling these opposing functions.

Disruption of the M80-Fe ligation stimulates the translocation of cytochrome c to the cytoplasm and nucleus in nonapoptotic cells

Native cytochrome c (cyt c) has a compact tertiary structure with a hexacoordinated heme iron and functions in electron transport in mitochondria and apoptosis in the cytoplasm. However, the possibility that protein modifications confer additional functions to cyt c has not been explored. Disruption of methionine 80 (M80)-Fe ligation of cyt c under nitrative stress has been reported. To model this alteration and determine if it confers new properties to cyt c, a cyt c mutant (M80A) was constitutively expressed in cells. M80A-cyt c has increased peroxidase activity and is spontaneously released from mitochondria, translocating to the cytoplasm and nucleus in the absence of apoptosis. Moreover, M80A models endogenously nitrated cyt c because nitration of WT-cyt c is associated with its translocation to the cytoplasm and nucleus. Further, M80A cyt c may up-regulate protective responses to nitrative stress. Our findings raise the possibility that endogenous protein modifications that disrupt the M80-Fe ligation (such as tyrosine nitration) stimulate nuclear translocation and confer new functions to cyt c in nonapoptotic cells.

Two pathways for tBID‐induced cytochrome c release from rat brain mitochondria: BAK‐ versus BAX‐dependence

The mechanisms of truncated BID (tBID)‐induced Cytu2003c release from non‐synaptosomal brain mitochondria were examined. Addition of tBID to mitochondria induced partial Cytu2003c release which was inhibited by anti‐BAK antibodies, implicating BAK. Immunoblotting showed the presence of BAK, but not BAX, in brain mitochondria. tBID did not release Cytu2003c from rat liver mitochondria, which lacked both BAX and BAK. This indicated that tBID did not act independently of BAX and BAK. tBID plus monomeric BAX produced twice as much Cytu2003c release as did tBID or oligomeric BAX alone. Neither tBID alone nor in combination with BAX induced mitochondrial swelling. In both cases Cytu2003c release was insensitive to cyclosporin A plus ADP, inhibitors of the mitochondrial permeability transition (mPT). Recombinant Bcl‐xL inhibited Cytu2003c release induced by tBID alone or in combination with monomeric BAX. Koenigs polyanion, an inhibitor of VDAC, suppressed tBID‐induced Cytu2003c release from brain mitochondria mediated by BAK but not by BAX. Thus, tBID can induce mPT‐independent Cytu2003c release from brain mitochondria by interacting with exogenous BAX and/or with endogenous BAK that may involve VDAC. In contrast, neither adenylate kinase nor Smac/DIABLO was released from isolated rat brain mitochondria via BAK or BAX.

Activation of calcium‐independent phospholipase A2 (iPLA2) in brain mitochondria and release of apoptogenic factors by BAX and truncated BID

Cleaved or truncated BID (tBID) is known to oligomerize both BAK and BAX. Previously, BAK and BAX lacing the C‐terminal fragment (BAXΔC) were shown to induce modest cytochrome c (Cytu2003c) release from rat brain mitochondria when activated by tBID. We now show that tBID plus monomeric full‐length BAX induce extensive release of Cytu2003c, Smac/DIABLO, and Omi/HtrA2 (but not endonuclease G and the apoptosis inducing factor) comparable to the release induced by alamethicin. This occurs independently of the permeability transition without overt changes in mitochondrial morphology. The mechanism of the release may involve formation of reactive oxygen species (ROS) and activation of calcium‐independent phospholipase A2 (iPLA2). Indeed, increased ROS production and activated iPLA2 were observed prior to massive Cytu2003c release. Furthermore, the extent of inhibition of Cytu2003c release correlated with the degree of suppression of iPLA2 by the inhibitors propranolol, dibucaine, 4‐bromophenacyl bromide, and bromenol lactone. Consistent with a requirement for iPLA2 in Cytu2003c release from brain mitochondria, synthetic liposomes composed of lipids mimicking the outer mitochondrial membrane (OMM) but lacing iPLA2 failed to release 10u2003kDa fluorescent dextran (FD‐10) in response to tBID plus BAX. We propose that tBID plus BAX activate ROS generation, which subsequently augments iPLA2 activity leading to changes in the OMM that allow translocation of certain mitochondrial proteins from the intermembrane space.

Leucine-rich alpha-2-glycoprotein-1 is upregulated in sera and tumors of ovarian cancer patients

BackgroundNew biomarkers that replace or are used in conjunction with the current ovarian cancer diagnostic antigen, CA125, are needed for detection of ovarian cancer in the presurgical setting, as well as for detection of disease recurrence. We previously demonstrated the upregulation of leucine-rich alpha-2-glycoprotein-1 (LRG1) in the sera of ovarian cancer patients compared to healthy women using quantitative mass spectrometry.MethodsLRG1 was quantified by ELISA in serum from two relatively large cohorts of women with ovarian cancer and benign gynecological disease. The expression of LRG1 in ovarian cancer tissues and cell lines was examined by gene microarray, reverse-transcriptase polymerase chain reaction (RT-PCR), Western blot, immunocytochemistry and mass spectrometry.ResultsMean serum LRG1 was higher in 58 ovarian cancer patients than in 56 healthy women (89.33 ± 77.90 vs. 42.99 ± 9.88 ug/ml; p = 0.0008) and was highest among stage III/IV patients. In a separate set of 193 pre-surgical samples, LRG1 was higher in patients with serous or clear cell ovarian cancer (145.82 ± 65.99 ug/ml) compared to patients with benign gynecological diseases (82.53 ± 76.67 ug/ml, p < 0.0001). CA125 and LRG1 levels were moderately correlated (r = 0.47, p < 0.0001). LRG1 mRNA levels were higher in ovarian cancer tissues and cell lines compared to their normal counterparts when analyzed by gene microarray and RT-PCR. LRG1 protein was detected in ovarian cancer tissue samples and cell lines by immunocytochemistry and Western blotting. Multiple iosforms of LRG1 were observed by Western blot and were shown to represent different glycosylation states by digestion with glycosidase. LRG1 protein was also detected in the conditioned media of ovarian cancer cell culture by ELISA, Western blotting, and mass spectrometry.ConclusionsSerum LRG1 was significantly elevated in women with ovarian cancer compared to healthy women and women with benign gynecological disease, and was only moderately correlated with CA125. Ovarian cancer cells secrete LRG1 and may contribute directly to the elevated levels of LRG1 observed in the serum of ovarian cancer patients. Future studies will determine whether LRG1 may serve as a biomarker for presurgical diagnosis, disease recurrence, and/or as a target for therapy.

ELISA for human serum leucine-rich alpha-2-glycoprotein-1 employing cytochrome c as the capturing ligand.

n Abstractn n Leucine-rich alpha-2-glycoprotein-1 (LRG) is a serum glycoprotein of unknown function that has shown promise based on qualitative assessments as a biomarker for certain diseases including microbial infections and cancer. However, the lack of a quantitative assay for LRG has limited its application. Here an indirect enzyme-linked immunosorbent assay (ELISA) for quantifying LRG in human serum is described in which cytochrome c is employed as the capturing ligand and a monoclonal antibody specific for LRG is used to detect the captured glycoprotein. Application of this assay in quantifying LRG in various patients sera is demonstrated. The concentration of LRG in sera of control subjects as determined by this assay is approximately 50xa0μg/ml. Consistent with expectations from published reports, LRG was found to be significantly elevated in the sera of some patients with a bacterial infection (toxic shock syndrome, TSS). LRG was only slightly elevated in patients infected with the human immunodeficiency virus as compared to uninfected control subjects, while normal levels of LRG were observed in patients with non-infectious diseases (inflammatory arthritis and neurological disorders, primarily Parkinsons disease). Although LRG and C-reactive protein (CRP) are both produced by the liver and are classified as acute-phase proteins, there was no significant correlation between the levels of LRG and CRP in the sera of the patients. Thus, LRG and CRP measurements are non-redundant and indicate different physiological contexts. The ELISA described in this report should be useful to further assess serum LRG as a biomarker for clinical diagnostics.n n

Affinity consideration in the design of synthetic vaccines intended to elicit antibodies

As a model for synthetic vaccines BALB/c mice were injected with a large cyanogen bromide cleaved fragment of horse cytochrome c, containing residues 1-80 of the 104 residue long polypeptide chain; then individual B cells specific for the peptide were challenged in vitro in splenic fragment cultures, with either the fragment or intact cytochrome c, both coupled to hemocyanin. The splenic environment in which the B cells were cultured contained hemocyanin-primed T cells, which provided equivalent T cell help for both the peptide and protein immunogens. In two experiments, intact cytochrome c-hemocyanin activated a total of only five peptide-primed B cells, compared to 66 that were activated by the peptide-hemocyanin conjugate. Furthermore, antibodies from the few cells that appeared to be activated by the protein did not bind the native protein with appreciable affinity in competitive ELISA. Of the mAbs elicited by the peptide, 51 were shown to have detectable affinity for native cytochrome c, but their affinity was dramatically less than that previously observed for antibodies elicited by protein-primed B cells and also less than that for the peptide. Thus, although the Ig receptors on many of the peptide-primed B cells did bind the protein to some extent, most such B cells were not activated. These results demonstrate that, in the development of synthetic vaccines, the affinity of a protein for peptide-primed antibodies (Ig receptors) is an important criterion to be considered. Qualitative examination of the binding of an anti-peptide antibody to a protein antigen, especially in denaturing conditions such as Western blots or in potentially denaturing conditions such as ELISA, is not an accurate indication of the efficacy of the peptide to prime B cells that can be activated upon challenge from the native protein.

Cytochrome c-induced lymphocyte death from the outside in: inhibition by serum leucine-rich alpha-2-glycoprotein-1

Previously we reported that serum leucine-rich alpha-2-glycoprotein-1 (LRG) binds cytochrome c (Cyt c; Cummings et al., Apoptosis 11:1121–1129, 2009). Here we show that LRG binding to Cyt c is similar to that of Apaf-1. LRG and Apaf-1 share partial amino acid sequences, compete for binding Cyt c, and are inhibited by modification at lysine 72 in Cyt c. However, in contrast to Apaf-1, LRG acts as a survival factor in vitro rather than a pro-apoptotic factor. By depleting LRG from culture medium we found that LRG protects against a toxic effect of exogenous Cyt c on lymphocytes that would otherwise result in an apoptotic phenotype. LRG, as well as antibodies specific for Cyt c, increased cell viability in the absence of added Cyt c indicating that Cyt c released by dying cells in the cultures is itself toxic. Protection from extracellular Cyt c-induced lymphotoxicity appears to involve an active mechanism rather than steric hindrance of Cyt c. Thus, serum LRG when bound to extracellular Cyt c that is released from apoptotic cells acts as a survival factor for lymphocytes and possibly other cells that are susceptible to the toxic effect of extracellular Cyt c.