Inhibition of Human Peptide Deformylase Disrupts Mitochondrial Function

Deformylases are metalloproteases in bacteria, plants, and humans that remove the N-formyl-methionine off peptides in vitro. The human homolog of peptide deformylase (HsPDF) resides in the mitochondria, along with its putative formylated substrates; however, the cellular function of HsPDF remains elusive. Here we report on the function of HsPDF in mitochondrial translation and oxidative phosphorylation complex biogenesis. Functional HsPDF appears to be necessary for the accumulation of mitochondrial DNA-encoded proteins and assembly of new respiratory complexes containing these proteins. Consequently, inhibition of HsPDF reduces respiratory function and cellular ATP levels, causing dependence on aerobic glycolysis for cell survival. A series of structurally different HsPDF inhibitors and control peptidase inhibitors confirmed that inhibition of HsPDF decreases mtDNA-encoded protein accumulation. Therefore, HsPDF appears to have a role in maintenance of mitochondrial respiratory function, and this function is analogous to that of chloroplast PDF.The human mitochondrial protein peptide deformylase, HsPDF, is a metalloprotease that removes the formyl moiety on the methionine of N-formyl-methionine peptide substrates in an enzymatic assay (24, 35). Despite the slow kinetic properties of HsPDF in an in vitro deformylation assay (24, 29, 35), we have shown that small interfering RNA (siRNA) interference of HsPDF decreases human cancer cell proliferation. Similarly, pharmacologic inhibition with the PDF antibiotic inhibitor actinonin and its analogs results in mitochondrial membrane depolarization and promotes cell death or proliferation arrest in a wide variety of cancer cell lines (18, 25). However, the cellular function of HsPDF remains elusive, and others have proposed that it has none (29). In bacteria, deformylation of nascent peptides is necessary for removal of the N-terminal methionine (36) and posttranslational processing of at least a subset of proteins that contribute to cell growth and viability (28). Prokaryotic PDF thus fulfills a role in cotranslational processing (7) and in protein degradation (41).In mammals, N-terminal formylation of proteins is only known to occur during mitochondrial translation initiation, as in prokaryotic protein translation (6). In contrast to bacteria, where the entire proteome is formylated for translation initiation, formylation in eukaryotes is limited to the 13 mitochondrial DNA (mtDNA)-encoded proteins. Formylation is important for mitochondrial translation, because formyl-Met-tRNA, but not Met-tRNA, is recognized by initiation factor 2 as the initiator tRNA (26, 37, 39). Therefore, the participation of HsPDF in protein post- or cotranslational processing can be narrowed down to these mitochondrial translation products.Despite the current understanding of the function of formyl-methionine in the initiation of protein synthesis in mammalian mitochondria (38, 39), the functional relevance of the downstream processing of nascent mitochondrial translation products has remained unexplored. Furthermore, it has been assumed that human mitochondria-encoded proteins, like those of bovine origin, are generally not deformylated after synthesis (45).The mammalian mitochondrial genome-encoded proteins are all subunits of four of the five oxidative phosphorylation respiratory chain enzyme complexes (I, III, IV, and V) (2, 40, 42). Respiratory complexes are comprised of multiple proteins. With the exception of complex II, which is comprised entirely of nuclear DNA-encoded subunits, all other complexes include both nuclear and mitochondrial DNA-encoded proteins. Synthesis of key mtDNA-encoded protein subunits, and the assembly of these proteins with multiple nuclear-encoded subunits within the mitochondria, is necessary for the function of each individual complex (16, 30, 44). Moreover, a functional interdependence among stably assembled respiratory complexes has been demonstrated (1). Mutations in human mtDNA that affect protein-coding regions or nuclear DNA mutations that affect expression of respiratory complex subunits cause disease (13), including Parkinson''s disease, for example, in which decreased respiratory function and compromised cell viability have been demonstrated (5, 21, 23). Therefore, the importance of properly assembled mitochondrial respiratory complexes suggests that their disruption, by inhibition of mtDNA-encoded protein processing, could have significant effects on cellular function.We hypothesized that HsPDF-mediated processing of mtDNA-encoded proteins is necessary for proper function of the respiratory chain complexes. To determine how the human deformylase activity contributes to cellular function, we used pharmacologic inhibition of HsPDF activity with the hydroxamic acid peptidomimetic inhibitor of PDF, actinonin, and confirmed our findings with a variety of other structurally different inhibitors. PDF has been shown to be a target of actinonin in bacteria (9), human cells (24), and plants (17).Here we show that inhibition of HsPDF function in mitochondria of human cell lines reduces mtDNA-encoded protein accumulation, new respiratory complex assembly, and energy production by the mitochondria. Aerobic glycolysis-dependent cell survival ensues upon disruption of HsPDF function. Therefore, HsPDF appears to fulfill a function in the mitochondria and to have a role in mtDNA-encoded protein-containing oxidative phosphorylation (OXPHOS) complex biogenesis.     

A new human peptide deformylase inhibitable by actinonin

Peptide deformylases (PDFs) have been investigated as potential specific targets for antibiotics, but the possible existence of a functional human PDF (HsPDF) presents a potential hurdle to the design of specific drugs. We have expression cloned a HsPDF that has deformylase activity, although it is a slower and catalytically less active enzyme than bacterial or plant PDFs. A cobalt-substituted form of HsPDF (but not nickel or zinc) is active, and the enzyme appears to be active at a pH between 6.0 and 7.2, a temperature range of 25-50 degrees C, and in a low KCl ionic strength buffer. Actinonin inhibits HsPDF activity with an IC50 of 43 nM and kills Daudi and HL60 human cancer cell lines with an LC50 of 5.3 and 8.8 microM, respectively. The inhibition of HsPDF may provide an explanation for the mechanism by which actinonin is cytotoxic against various human tumor cell lines.     

Structure and Activity of Human Mitochondrial Peptide Deformylase, a Novel Cancer Target

Peptide deformylase proteins (PDFs) participate in the N-terminal methionine excision pathway of newly synthesized peptides. We show that the human PDF (HsPDF) can deformylate its putative substrates derived from mitochondrial DNA-encoded proteins. The first structural model of a mammalian PDF (1.7 Å), HsPDF, shows a dimer with conserved topology of the catalytic residues and fold as non-mammalian PDFs. The HsPDF C-terminus topology and the presence of a helical loop (H2 and H3), however, shape a characteristic active site entrance. The structure of HsPDF bound to the peptidomimetic inhibitor actinonin (1.7 Å) identified the substrate-binding site. A defined S1′ pocket, but no S2′ or S3′ substrate-binding pockets, exists. A conservation of PDF-actinonin interaction across PDFs was observed. Despite the lack of true S2′ and S3′ binding pockets, confirmed through peptide binding modeling, enzyme kinetics suggest a combined contribution from P2′and P3′ positions of a formylated peptide substrate to turnover.     

High-throughput identification of inhibitors of human mitochondrial peptide deformylase

The human mitochondrial peptide deformylase (HsPDF) provides a potential new target for broadly acting antiproliferative agents. To identify novel nonpeptidomimetic and nonhydroxamic acid-based inhibitors of HsPDF, the authors have developed a high-throughput screening (HTS) strategy using a fluorescence polarization (FP)-based binding assay as the primary assay for screening chemical libraries, followed by an enzymatic-based assay to confirm hits, prior to characterization of their antiproliferative activity against established tumor cell lines. The authors present the results and performance of the established strategy tested in a pilot screen of 2880 compounds and the identification of the 1st inhibitors. Two common scaffolds were identified within the hits. Furthermore, cytotoxicity studies revealed that most of the confirmed hits have antiproliferative activity. These findings demonstrate that the designed strategy can identify novel functional inhibitors and provide a powerful alternative to the use of functional assays in HTS and support the hypothesis that HsPDF inhibitors may constitute a new class of antiproliferative agent.     

Plant peptide deformylase: a novel selectable marker and herbicide target based on essential cotranslational chloroplast protein processing

Transgenic tobacco plants expressing three different forms of Arabidopsis plant peptide deformylase ( At DEF1.1, At DEF1.2 and At DEF2; EC 3.5.1.88) were evaluated for resistance to actinonin, a naturally occurring peptide deformylase inhibitor. Over-expression of either AtDEF1.2 or AtDEF2 resulted in resistance to actinonin, but over-expression of AtDEF1.1 did not. Immunological analyses demonstrated that At DEF1.2 and At DEF2 enzymes were present in both stromal and thylakoid fractions in chloroplasts, but At DEF1.1 was localized to mitochondria. The highest enzyme activity was associated with stromal At DEF2, which was approximately 180-fold greater than the level of endogenous activity in the host plant. Resistance to actinonin cosegregated with kanamycin resistance in Atdef1.2-D and Atdef2-D transgenic plants. Here, we demonstrate that the combination of plant peptide deformylase and peptide deformylase inhibitors may represent a native gene selectable marker system for chloroplast and nuclear transformation vectors, and also suggest plant peptide deformylase as a potential broad-spectrum herbicide target.     

Identification of benzofuran-4,5-diones as novel and selective non-hydroxamic acid, non-peptidomimetic based inhibitors of human peptide deformylase

Selective inhibitors of human peptide deformylase (HsPDF) are predicted to constitute a new class of antitumor agents. We report the identification of benzofuran-4,5-diones as the first known selective HsPDF inhibitors and we describe their selectivity profile in a panel of metalloproteases. We characterize their structure-activity relationships for antitumor activity in a panel of cancer cell lines, and we assess their in vivo efficacy in a mouse xenograft model. Our results demonstrate that selective HsPDF inhibitors based on the benzofuran-4,5-dione scaffold constitute a novel class of antitumor agents that are potent in vitro and in vivo.     

The role of peptide deformylase in protein biosynthesis: a proteomic study

Recently we investigated the influence of classical and emerging antibiotics on the proteome of Bacillus subtilis including in our studies actinonin, a potent novel inhibitor of peptide deformylase. The protein synthesis pattern under actinonin treatment changed so dramatically that a direct comparison to the control pattern was impossible. Dual channel imaging revealed that actinonin treatment caused the majority of newly synthesised proteins to accumulate in spots different from the ones usually observed, indicating a more acidic isoelectric point. Two strategies were used to investigate the nature of the charge shift. In the first place, protein patterns of a conditional peptide deformylase mutant under nonrepressing and repressing conditions were compared. Secondly, several protein pairs excised from two-dimensional (2-D) gels of the peptide deformylase mutant, exponentially growing untreated wild-type and the actinonin treated wild-type were investigated with matrix-assisted laser desorption/ionization and electrospray ionization (ESI) time of flight mass spectrometry (TOF MS) for the existence of N-terminal formylation. Under nonrepressing conditions the mutant protein pattern resembled that of the wild-type. The loss of peptide deformylase activity under repressing conditions led to the same pI shift observed for actinonin treatment in the wild-type. Quadrupole TOF-MS on 11 protein pairs proved that the remaining N-terminal formyl residue was indeed responsible for the charge shift. Eight of these protein pairs were also present on 2-D gels of exponentially growing B. subtilis, where the more acidic, still formylated protein species represented the smaller parts.     

Over-expression of peptide deformylase in chloroplasts confers actinonin resistance,but is not a suitable selective marker system for plastid transformation

Arabidopsis thaliana peptide deformylase PDF1B was expressed in tobacco chloroplasts using spectinomycin as the selective agent. The foreign protein accumulated in chloroplasts (6% of the total soluble protein) and was enzymatically active. Transplastomic plants were evaluated for resistance to the peptide deformylase inhibitor actinonin. In vitro seed germination in the presence of actinonin and in planta application of the inhibitor demonstrated the resistance of the transformed plants. In addition, transgenic leaf explants were able to develop shoots via organogenesis in the presence of actinonin. However, when the combination of the PDF1B gene and actinonin was used as the primary selective marker system for chloroplast transformation of tobacco, all developed shoots were escapes. Therefore, under the experimental conditions tested, the use of this system for plastid transformation would be limited to function as a secondary selective marker.     

Characterization of a human peptide deformylase: implications for antibacterial drug design

Ribosomal protein synthesis in eubacteria and eukaryotic organelles initiates with an N-formylmethionyl-tRNA(i), resulting in N-terminal formylation of all nascent polypeptides. Peptide deformylase (PDF) catalyzes the subsequent removal of the N-terminal formyl group from the majority of bacterial proteins. Deformylation was for a long time thought to be a feature unique to the prokaryotes, making PDF an attractive target for designing novel antibiotics. However, recent genomic sequencing has revealed PDF-like sequences in many eukaryotes, including man. In this work, the cDNA encoding Homo sapiens PDF (HsPDF) has been cloned and a truncated form that lacks the N-terminal 58-amino-acid targeting sequence was overexpressed in Escherichia coli. The recombinant, Co(2+)-substituted protein is catalytically active in deformylating N-formylated peptides, shares many of the properties of bacterial PDF, and is strongly inhibited by specific PDF inhibitors. Expression of HsPDF fused to the enhanced green fluorescence protein in human embryonic kidney cells revealed its location in the mitochondrion. However, HsPDF is much less active than its bacterial counterpart, providing a possible explanation for the apparent lack of deformylation in the mammalian mitochondria. The lower catalytic activity is at least partially due to mutation of a highly conserved residue (Leu-91 in E. coli PDF) in mammalian PDF. PDF inhibitors had no detectable effect on two different human cell lines. These results suggest that HsPDF is likely an evolutional remnant without any functional role in protein formylation/deformylation and validates PDF as an excellent target for antibacterial drug design.     

Eukaryotic peptide deformylases. Nuclear-encoded and chloroplast-targeted enzymes in Arabidopsis

Arabidopsis (ecotype Columbia-0) genes, AtDEF1 and AtDEF2, represent eukaryotic homologs of the essential prokaryotic gene encoding peptide deformylase. Both deduced proteins contain three conserved protein motifs found in the active site of all eubacterial peptide deformylases, and N-terminal extensions identifiable as chloroplast-targeting sequences. Radiolabeled full-length AtDEF1 was imported and processed by isolated pea (Pisum sativum L. Laxton's Progress No. 9) chloroplasts and AtDEF1 and 2 were immunologically detected in Arabidopsis leaf and chloroplast stromal protein extracts. The partial cDNAs encoding the processed forms of Arabidopsis peptide deformylase 1 and 2 (pAtDEF1 and 2, respectively) were expressed in Escherichia coli and purified using C-terminal hexahistidyl tags. Both recombinant Arabidopsis peptide deformylases had peptide deformylase activity with unique kinetic parameters that differed from those reported for the E. coli enzyme. Actinonin, a specific peptide deformylase inhibitor, was effective in vitro against Arabidopsis peptide deformylase 1 and 2 activity, respectively. Exposure of several plant species including Arabidopsis to actinonin resulted in chlorosis and severe reductions in plant growth and development. The results suggest an essential role for peptide deformylase in protein processing in all plant plastids.     

Inhibition and structure-activity studies of methionine hydroxamic acid derivatives with bacterial peptide deformylase

The posttranslational deformylation of N-formyl-Met-polypeptides by the metalloenzyme, peptide deformylase, is essential for bacterial growth. Methionine hydroxamic acid derivatives were found to inhibit recombinant Escherichia coli peptide deformylase activity containing either zinc or cobalt. The binding of methionine hydroxamate and hydrazide inhibitors to cobalt-substituted deformylase caused spectral changes consistent with the formation of a pentacoordinate metal complex similar to that of actinonin, a psuedopeptide hydroxamate inhibitor. The spectral and kinetic data support the binding of these N-substituted L-methionine derivatives in a reverse orientation with respect to N-formyl-Met-peptide substrates within the active site. Based on this hypothesis a second generation of N-substituted methionyl hydroxamic acids were evaluated and found to possess greater inhibitory potency. These results may provide the basis for the design of more potent and selective deformylase inhibitors as potential antibacterial agents.     

Death-associated protein kinase as a sensor of mitochondrial membrane potential: role of lysosome in mitochondrial toxin-induced cell death

We have investigated here the mechanism of dephosphorylation and activation of death-associated protein kinase (DAPK) and the role of lysosome in neuroblastoma cells (SH-SY5Y) treated with mitochondrial toxins, such as MPP(+) and rotenone. Mitochondrial respiratory chain inhibitors and uncouplers decreased mitochondrial membrane potential leading to DAPK dephosphorylation and activation. The class III phosphoinositide 3-kinase inhibitors attenuated DAPK dephosphorylation induced by mitochondrial toxins. Complex I inhibition by mitochondrial toxins (e.g. MPP(+)) resulted in mitochondrial swelling and lysosome reduction. Inhibition of class III phosphoinositide 3-kinase attenuated MPP(+)-induced lysosome reduction and cell death. The role of DAPK as a sensor of mitochondrial membrane potential in mitochondrial diseases was addressed.     

Surface Levels of CD20 Determine Anti-CD20 Antibodies Mediated Cell Death In Vitro

Background

The sensitivity of human Burkitt''s lymphoma cells to rituximab (Rtx) and tositumomab (Tst) was assessed on cells expressing different levels of CD20 on surface. Cells that harbor low CD20 levels may resists against therapeutics response to CD20-specific antibodies. We postulated that, radiation-induced modulation of CD20 surface levels may play a crucial and central role in determining the relative efficacy of rituximab and tositumomab in treating Burkitt''s lymphoma disease. Here, we examined the γ-radiation-induced CD20 expression in the Burkitt lymphoma cell line ‘Daudi’ and the relation of differential levels of CD20 with anti-CD20 mAbs mediated cell death.

Methodology

In this study we examined kinetics of CD20 expression following sub lethal doses ofγ-radiation to Daudi cells and thereafter anti-CD20 mAbs (rituximab and tositumomab) were added in cell suspensions. The correlation of kinetics of CD20 expression and cells treated with anti-CD20 mAbs/or corresponding isotype Abs with special reference to changes in mitochondrial membrane potential and reactive oxygen species generation was also examined. Further, we also investigated the efficacy of anti-CD20 mAbs and possible induction of cell death in relation to levels of CD20 cell surface expression.

Conclusion

This report provides evidence that CD20 expression can be induced by exposure of cells to γ-radiation. In addition, these findings demonstrated that the efficacy of anti-CD20 mAbs is dependent on the surface levels of CD20. Based on these findings, we hypothesized (i) irradiation just prior to immunotherapy may provide new treatment options even in aggressive B cell tumors, which are resistant to current therapies in vivo (ii) The efficacy of induction of apoptosis varies with type of monoclonal antibodies in vitro.     

B cell receptor cross-linking triggers a caspase-8-dependent apoptotic pathway that is independent of the death effector domain of Fas-associated death domain protein.

We have previously reported that B cell receptors, depending on the degree to which they are cross-linked, can promote apoptosis in various human B cell types. In this study, we show that B cell receptors can trigger two apoptotic pathways according to cross-linking and that these pathways control mitochondrial activation in human Burkitt's lymphoma cells. Whereas soluble anti-mu Ab triggers caspase-independent mitochondrial activation, cross-linked anti-mu Ab induces an apoptotic response associated with a caspase-dependent loss of mitochondrial transmembrane potential. This B cell receptor-mediated caspase-dependent mitochondrial activation is associated with caspase-8 activation. We show here that caspase-8 inhibitors strongly decrease cross-linking-dependent B cell receptor-mediated apoptosis in Burkitt's lymphoma BL41 cells. These inhibitors act upstream from the mitochondria as they prevented the loss of mitochondrial membrane potential observed in B cell receptor-treated BL41 cells. Caspase-8 activation in these cells was also evident from the detection of cleaved fragments of caspase-8 and the cleavage of specific substrates, including Bid. Our data show that cross-linked B cell receptors induced an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and the activation of caspase-9 and caspase-3. Cells expressing a dominant negative mutant of Fas-associated death domain protein were sensitive to cross-linked B cell receptor-induced caspase-8 activation and apoptosis; therefore, this caspase-8 activation was independent of the death effector domain of Fas-associated death domain protein.     

1H, 13C and 15N NMR assignments of the E. coli peptide deformylase in complex with a natural inhibitor called actinonin

In eubacteria, the formyl group of nascent polypeptides is removed by peptide deformylase protein (PDF). This is the reason why PDF has received special attention in the course of the search for new antibacterial agents. We observed by NMR that actinonin, a natural inhibitor, induced drastic changes in the HSQC spectrum of E. coli PDF. We report here the complete NMR chemical shift assignments of PDF resonances bound to actinonin.     

Single agent and synergistic combinatorial efficacy of first-in-class small molecule imipridone ONC201 in hematological malignancies

ONC201, founding member of the imipridone class of small molecules, is currently being evaluated in advancer cancer clinical trials. We explored single agent and combinatorial efficacy of ONC201 in preclinical models of hematological malignancies. ONC201 demonstrated (GI50 1–8 µM) dose- and time-dependent efficacy in acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Burkitt's lymphoma, anaplastic large cell lymphoma (ALCL), cutaneous T-cell lymphoma (CTCL), Hodgkin's lymphoma (nodular sclerosis) and multiple myeloma (MM) cell lines including cells resistant to standard of care (dexamethasone in MM) and primary samples. ONC201 induced caspase-dependent apoptosis that involved activation of the integrated stress response (ATF4/CHOP) pathway, inhibition of Akt phosphorylation, Foxo3a activation, downregulation of cyclin D1, IAP and Bcl-2 family members. ONC201 synergistically reduced cell viability in combination with cytarabine and 5-azacytidine in AML cells. ONC201 combined with cytarabine in a Burkitt's lymphoma xenograft model induced tumor growth inhibition that was superior to either agent alone. ONC201 synergistically combined with bortezomib in MM, MCL and ALCL cells and with ixazomib or dexamethasone in MM cells. ONC201 combined with bortezomib in a Burkitt's lymphoma xenograft model reduced tumor cell density and improved CHOP induction compared to either agent alone. These results serve as a rationale for ONC201 single-agent trials in relapsed/refractory acute leukemia, non-Hodgkin's lymphoma, MM and combination trial with dexamethasone in MM, provide pharmacodynamic biomarkers and identify further synergistic combinatorial regimens that can be explored in the clinic.     

Inhibition of peptide deformylase in Nicotiana tabacum leads to decreased D1 protein accumulation, ultimately resulting in a reduction of photosystem II complexes

Eukaryotic homologs of bacterial peptide deformylases were recently found in several vascular plants and may be essential in chloroplast protein processing. Treating tobacco seedlings with the peptide deformylase inhibitor actinonin resulted in leaf chlorosis and reduced growth and development, indicative of a systemic movement of the inhibitor. Photosystem II (PSII) activity was reduced, manifested as a significant decrease in the maximum quantum efficiency of photosystem II. Accumulation and assembly of nascent D1 protein into PSII monomers was also reduced, eventually leading to PSII disassembly and leaf necrosis. Processing and assembly of D1 protein in tobacco was a major and potentially critical target of peptide deformylase inhibition. These results confirm that N-terminal deformylation is an essential step in the accumulation and assembly of PSII subunit polypeptides in the chloroplasts of vascular plants.     

Ribosome inactivating protein saporin induces apoptosis through mitochondrial cascade, independent of translation inhibition

Ribosome inactivating proteins (RIPs) are toxic translation inhibitors that kill eukaryotic cells by arresting protein synthesis at the translocation step. Saporin-6, expressed in the seeds of Saponaria officinalis plant, is a type I RIP comprising of a single polypeptide chain. Saporin is a specific RNA N-glycosidase and it removes a specific adenine residue from a conserved loop of the large rRNA of eukaryotic cells. Saporin-6 is one of the most potent of several isoforms of saporin, obtained from different tissues of the Saponaria plant. In addition to potently inhibiting translation, saporin has been also shown to induce cell death by apoptosis in different cellular models. To elucidate the mechanism of apoptosis induction by saporin, we have investigated the apoptotic pathway triggered by saporin. We have also analyzed whether the inhibition of protein synthesis by the toxin is the trigger for induction of apoptosis. We demonstrate that saporin-6 induces caspase-dependent apoptosis in U937 cells via the mitochondrial or intrinsic pathway. Unlike many other toxins the catalytic N-glycosidase activity of saporin is not required for apoptosis induction, and the apoptosis onset occurs before any significant inhibition of protein synthesis ensues.     

Immobilized α2,6-linked sialic acid suppresses caspase-3 activation during anti-IgM antibody-induced apoptosis in Ramos cells

In Ramos cells, a human Burkitt's lymphoma cell line, stimulation of the B cell antigen receptor with anti-IgM antibody (Ab) induces apoptosis as indicated by a decrease in cell viability and an increase in DNA fragmentation and cell surface exposure of phosphatidylserine. Furthermore, these changes are suppressed by incubating the cells in α₁-acid glycoprotein (AGP)-coated tissue culture plates. Here, we found that, during Anti-IgM Ab-induced apoptosis in Ramos cells, caspase-3 is activated downstream of caspase-8 and the mitochondrial pathway is activated, as indicated by a loss of mitochondrial membrane potential, an increase in the release of cytochrome c to the cytoplasm, and enhanced Bax expression. Anti-IgM Ab-induced apoptosis of neuraminidase-treated Ramos cells was suppressed by incubating the cells on plates coated with AGP, which contains a high concentration of α2,6-linked sialic acid. The incubation on plates coated with AGP also suppressed anti-IgM Ab-stimulated caspase-3 activity and increased the level of X-linked inhibitor of apoptosis protein (XIAP), but it did not affect caspase-8 activity, the mitochondrial membrane potential, cytochrome c release, or Bax expression. The results indicate that the interaction of Ramos cells with immobilized α2,6-linked sialic acid enhances XIAP expression, directly or indirectly suppressing caspase-3 activity and inhibiting anti-IgM Ab-induced apoptosis.     

Activation of NFκB and Ub-proteasome pathway during apoptosis induced by a serum factor is mediated through the upregulation of the 26S proteasome subunits

We have been investigating differential gene expression associated with apoptosis in AK-5 cells (a spontaneously regressing rat histiocytoma) and have observed catalytic subunits beta 7 and alpha 5 of the 26S proteasome and ubiquitin to be upregulated during apoptosis induced by a variety of agents. The observed elevation in gene expression was parallel to a comparable increase in the cytosolic protein expression of the proteasome and ubiquitin and a markedly amplified increase in the proteasome activity. Inhibition of the increase in gene expression resulted in the inhibition of the rise in the proteasome activity subsequently leading to an inhibition of apoptosis. Similarly, pretreatment with proteasome inhibitors, MG132 and lactacystin, resulted in a significant inhibition of apoptosis pointing to the requirement of a highly active protein degradation machinery during apoptosis. The apoptosis inhibitory effect of the proteasome inhibitors involved an inhibition of the activation of various initiator and effector caspases but was independent of any changes in the mitochondrial membrane depolarization and cytochrome c release associated with apoptosis. Inhibition of proteasome activity or its upstream PI3 kinase activity inhibited NFκB translocation thereby suppressing apoptosis, which highlights the requirement of NFκB activation for completion of apoptosis in AK-5 cells. Hence, the apoptosis associated induction of the Ub-proteasome pathway components and the proteasome activity suggests that the proteasome, in its capacity as an efficient protein degradation complex, plays an important role in the successful execution of apoptosis.