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PubMed1984

Cytochrome c mediates electron transfer between ubiquinol-cytochrome c reductase and cytochrome c oxidase by free diffusion along the surface of the membrane.

Ubiquinol oxidase can be reconstituted from ubiquinol-cytochrome c reductase (Complex III) and cytochrome c oxidase (Complex IV) whose endogenous phosphatidylcholine and phosphatidylethanolamine have been replaced by dimyristoylglycerophosphocholine. Phase transition of the lipid has no effect on Complex III and Complex IV activities assayed separately, but ubiquinol oxidase activity rapidly decreases as the temperature is lowered through the phase transition. A spin-labelled yeast cytochrome c derivative has been synthesized. Binding of the cytochrome c to liposomes demonstrates that only cardiolipin is involved under the conditions used for the ubiquinol oxidase experiments. In liposomes consisting of cardiolipin and dimyristoylglycerophosphocholine, e.s.r. (electron-spin-resonance) measurements show that rotational diffusion of cytochrome c is slowed in the gel phase of the latter lipid. We propose that the cytochrome c pool is bound to cardiolipin molecules, whose lateral and rotational diffusion in the bilayer is adequate to account for electron-transport rates.

Cell Membrane↗
PubMed1997

Double-cleavage production of the CTL epitope by proteasomes and PA28: role of the flanking region.

BACKGROUND: Proteasomes are known to produce major histocompatibility complex (MHC) class I ligands from endogenous antigens, and the gamma-interferon-inducible proteasome activator PA28 has been thought to play an important role in the generation of immunodominant MHC ligands by proteasomes. Several attempts have been made to show that proteasomes have the ability to yield cytotoxic T lymphocyte (CTL) epitopes effectively from model polypeptides derived from viral and intracellular proteins in vitro, but their antigen processing mechanism is poorly understood. RESULTS: Proteasomes produce the tumour rejection antigen precursor peptide pRL1b (SIIPGLPLSL), but not pRL1a (IPGLPLSL), bound to the H-2Ld molecule, from synthetic peptides covering the CTL epitope. This double cleavage production of pRL1b by proteasomes seemed to depend on the length of the flanking regions adjacent to either end of the CTL epitope, in which their successive deletions caused the almost complete prevention of pRL1b excision. The newly identified PA28 collaborates with proteasomes for efficient production of pRL1b, by promoting not only single cleavage of all susceptible peptides, but also dual cleavage in some peptides harboring certain characteristic lengths. CONCLUSION: The flanking regions outside pRL1b of suitable length appear to be essential for the correct CTL epitope production, possibly functioning as anchors to trap target peptides for proteasomal degradation. We propose a novel mechanism for dual-cleavage excision of immunodominant epitopes by proteasomes and PA28.

Amino Acid Sequence↗
PubMed2000

Astrocyte-derived nitric oxide causes both reversible and irreversible damage to the neuronal mitochondrial respiratory chain.

Cytokine-stimulated astrocytes produce nitric oxide (NO), which, along with its metabolite peroxynitrite (ONOO(-)), can inhibit components of the mitochondrial respiratory chain. We used astrocytes as a source of NO/ONOO(-) and monitored the effects on neurons in coculture. We previously demonstrated that astrocytic NO/ONOO(-) causes significant damage to the activities of complexes II/III and IV of neighbouring neurons after a 24-h coculture. Under these conditions, no neuronal death was observed. Using polytetrafluoroethane filters, which are permeable to gases such as NO but impermeable to NO derivatives, we have now demonstrated that astrocyte-derived NO is responsible for the damage observed in our coculture system. Expanding on these observations, we have now shown that 24 h after removal of NO-producing astrocytes, neurons exhibit complete recovery of complex II/III and IV activities. Furthermore, extending the period of exposure of neurons to NO-producing astrocytes does not cause further damage to the neuronal mitochondrial respiratory chain. However, whereas the activity of complex II/III recovers with time, the damage to complex IV caused by a 48-h coculture with NO-producing astrocytes is irreversible. Therefore, it appears that neurons can recover from short-term damage to mitochondrial complex II/III and IV, whereas exposure to astrocytic-derived NO for longer periods causes permanent damage to neuronal complex IV.

Animals↗
PubMed1992

Proteasomes are regulated by interferon gamma: implications for antigen processing.

Class I major histocompatibility complex (MHC) molecules present antigenic peptides of cytoplasmic origin to T cells. As the lengths of these peptides seem restricted to eight or nine amino acids, an unusual proteolytic system must play a role in antigen processing. Proteasomes, a major extralysosomal proteolytic system, are responsible for the degradation of cytoplasmic proteins. We demonstrate that several proteasomal subunits, including MHC-encoded subunits, are regulated by interferon gamma. These data and the finding that MHC-encoded and other interferon gamma-regulated proteasomal subunits are uniquely associated with proteasomes strongly suggest that the immune system has recruited proteasomes for antigen processing.

Animals↗
PubMed1992

Genomic polymorphism, recombination, and linkage disequilibrium in human major histocompatibility complex-encoded antigen-processing genes.

Recently, two subunits of a large cytosolic protease and two putative peptide transporter proteins were found to be encoded by genes within the class II region of the major histocompatibility complex (MHC). These genes have been suggested to be involved in the processing of antigenic proteins for presentation by MHC class I molecules. Because of the high degree of polymorphism in MHC genes, and previous evidence for both functional and polypeptide sequence polymorphism in the proteins encoded by the antigen-processing genes, we tested DNA from 27 consanguineous human cell lines for genomic polymorphism by restriction fragment length polymorphism (RFLP) analysis. These studies demonstrate a strong linkage disequilibrium between TAP1 and LMP2 RFLPs. Moreover, RFLPs, as well as a polymorphic stop codon in the telomeric TAP2 gene, appear to be in linkage disequilibrium with HLA-DR alleles and RFLPs in the HLA-DO gene. A high rate of recombination, however, seems to occur in the center of the complex, between the TAP1 and TAP2 genes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Peptidase activities of proteasomes are differentially regulated by the major histocompatibility complex-encoded genes for LMP2 and LMP7.

Recent studies have implicated proteasomes in the generation of the antigenic peptides that are presented on major histocompatibility complex class I molecules to T lymphocytes. Interferon gamma modifies the subunit composition of proteasomes and causes changes in their peptidase activities that should favor the production of peptides with hydrophobic or basic carboxyl termini (i.e., the types found on major histocompatibility complex class I molecules). It has been proposed that these changes in peptidase activity are due to incorporation into proteasomes of the major histocompatibility complex-encoded subunits LMP2 and -7, which are induced by interferon gamma. Here we show by gene transfection into lymphoblasts or HeLa cells that LMP7 increases the capacity (Vmax) of 20S and 26S proteasomes to cleave peptides after hydrophobic and basic residues without affecting hydrolysis after acidic residues. These changes depended on the amount of LMP7 subunits incorporated into proteasomes. Transfection of LMP2 reduced cleavage of peptides after acidic residues, increased hydrolysis after basic residues, and did not affect the hydrophobic activity. Since the activity of the total proteasome population changed after incorporation of only small amounts of LMP2 or -7, these subunits must cause major alterations in peptidase activity. Thus, their expression can account for the changes in proteasome activity induced by inteferon gamma, and these findings lend further support to the proposed roles of LMPs in altering the nature of the peptides generated for antigen presentation.

Amino Acid Sequence↗
PubMed1995

Effects of interferon gamma and major histocompatibility complex-encoded subunits on peptidase activities of human multicatalytic proteases.

We have examined several peptidase activities of human multicatalytic protease (MCP) purified from the lymphoblastoid cell line 721.45 and a deletion mutant derivative, 721.174, lacking MCP subunits encoded in the major histocompatibility complex (MHC) class II region. Wild-type lymphoblast MCP hydrolyzed a specific peptide, glutaryl-Gly-Gly-Phe-4-methylcoumaryl-7-amide (-MCA), several times faster than the mutant enzyme did, suggesting that MHC-encoded subunits may provide this activity. Contrary to a recent report [Driscoll, J., Brown, M. G., Finley, D. & Monaco, J J. (1993) Nature (London) 365, 262-264], we did not detect significant aminopeptidase associated with lymphoblast MCPs. Our results also differ markedly from those of Gaczynska et al. [Gaczynska, M., Rock, K. L. & Goldberg, A L. (1993) Nature (London) 365, 264-267], who reported that gamma interferon (IFN-gamma) alters the peptidase activities of lymphoblast MCPs. We found that IFN-gamma did not produce significant differences in the peptidase activities of purified MCPs. Moreover, our measurements of Vmax and Km for succinyl-Leu-Leu-Val-Tyr-MCA hydrolysis differ 600-fold and 15-fold, respectively, from those reported by Gaczynska et al. On balance, the findings presented here do not support the idea that IFN-gamma induces major changes in the peptidase activity of purified MCPs.

Amino Acid Sequence↗
PubMed1995

Major histocompatibility-encoded human proteasome LMP2. Genomic organization and a new form of mRNA.

LMP2 is one of the two proteasome subunits encoded by genes in the major histocompatibility complex class II region. Here we report the genomic organization of human LMP2 gene. Sequence analysis of polymerase chain reaction-amplified cDNA from a number of lymphoblastoid cell lines demonstrated two forms of LMP2 mRNA, one (LMP2.1) complete and homologous to the published LMP2 genomic sequence from cosmid clones, and the other (LMP2.s) a smaller transcript resulting from splicing of a 30-base pair fragment from the first exon. Antibodies to recombinant LMP2.s protein (22.3 kDa) were raised in rabbits. This anti-LMP2.s serum recognized both recombinant proteins (LMP2.1 = 23.3 kDa and LMP2.s = 22.3 kDa) and a single protein of 21.5 kDa molecular mass in lysates from human lymphoblastoid cell lines. Pulse-chase experiments demonstrated that LMP2 polypeptide also undergoes processing from 22.3- to 21.5-kDa protein when incorporated into proteasomes. These data suggest that the processing of human LMP2 subunit takes place both at the transcription and post-translational levels. Northern blot analysis showed that the LMP2 mRNA is expressed in lymphoblastoid cell lines and in fibroblasts following gamma-interferon induction, but not in brain, smooth muscle, fibroblasts (uninduced), and colon epithelial cells.

Amino Acid Sequence↗
PubMed1995

In vivo assembly of the proteasomal complexes, implications for antigen processing.

The multicatalytic and multisubunit proteasomal complexes have been implicated in the processing of antigens to peptides presented by class I major histocompatibility complex molecules. Two structural complexes of this proteinase, 20 S and 26 S proteasomes, have been isolated from cells. By analyzing in vivo assembly of the proteasomal complexes we show that the 20 S proteasomal complexes are irreversibly assembled via 15 S assembly intermediates containing unprocessed beta-type subunits. The 20 S proteasomes further associate reversibly with proteasome activators PA28 or pre-existing ATPase complexes to form 26 S proteasomal complexes. Our findings that not all of the 20 S proteasomal complexes are assembled into 26 S proteasomal complexes within cells and that all of PA28 and ATPase complexes are associated with 20 S proteasomes strongly suggest that all proteasomal complexes coexist within cells. We further demonstrate that 26 S proteasomal complexes are predominantly present in the cytoplasm and a significant portion of the 20 S proteasomal complexes is associated with the endoplasmic reticulum membrane. Taken together, our findings suggest that depending upon their associated regulatory components, 26 S and 20 S-PA28 proteasomal complexes serve different housekeeping functions within the cells, while they degrade antigens in a cooperative manner in antigen processing.

Animals↗
PubMed1995

Rhodoquinone and complex II of the electron transport chain in anaerobically functioning eukaryotes.

Many anaerobically functioning eukaryotes have an anaerobic energy metabolism in which fumarate is reduced to succinate. This reduction of fumarate is the opposite reaction to succinate oxidation catalyzed by succinate-ubiquinone oxidoreductase, complex II of the aerobic respiratory chain. Prokaryotes are known to contain two distinct enzyme complexes and distinct quinones, menaquinone and ubiquinone (Q), for the reduction of fumarate and the oxidation of succinate, respectively. Parasitic helminths are also known to contain two different quinones, Q and rhodoquinone (RQ). This report demonstrates that RQ was present in all examined eukaryotes that reduce fumarate during anoxia, not only in parasitic helminths, but also in freshwater snails, mussels, lugworms, and oysters. It was shown that the measured RQ/Q ratio correlated with the importance of fumarate reduction in vivo. This is the first demonstration of the role of RQ in eukaryotes, other than parasitic helminths. Furthermore, throughout the development of the liver fluke Fasciola hepatica, a strong correlation was found between the quinone composition and the type of metabolism: the amount of Q was correlated with the use of the aerobic respiratory chain, and the amount of RQ with the use of fumarate reduction. It can be concluded that RQ is an essential component for fumarate reduction in eukaryotes, in contrast to prokaryotes, which use menaquinone in this process. Analyses of enzyme kinetics, as well as the known differences in primary structures of prokaryotic and eukaryotic complexes that reduce fumarate, support the idea that fumarate-reducing eukaryotes possess an enzyme complex for the reduction of fumarate, structurally related to the succinate dehydrogenase-type complex II, but with the functional characteristics of the prokaryotic fumarate reductases.

Animals↗
PubMed1999

An Escherichia coli mutant quinol:fumarate reductase contains an EPR-detectable semiquinone stabilized at the proximal quinone-binding site.

The EPR and thermodynamic properties of semiquinone (SQ) species stabilized by mammalian succinate:quinone reductase (SQR) in situ in the mitochondrial membrane and in the isolated enzyme have been well documented. The equivalent semiquinones in bacterial membranes have not yet been characterized, either in SQR or quinol:fumarate reductase (QFR) in situ. In this work, we describe an EPR-detectable QFR semiquinone using Escherichia coli mutant QFR (FrdC E29L) and the wild-type enzyme. The SQ exhibits a g = 2.005 signal with a peak-to-peak line width of approximately 1.1 milliteslas at 150 K, has a midpoint potential (E(m(pH 7.2))) of -56.6 mV, and has a stability constant of approximately 1.2 x 10(-2) at pH 7.2. It shows extremely fast spin relaxation behavior with a P(1/2) value of >>500 milliwatts at 150 K, which closely resembles the previously described SQ species (SQ(s)) in mitochondrial SQR. This SQ species seems to be present also in wild-type QFR, but its stability constant is much lower, and its signal intensity is near the EPR detection limit around neutral pH. In contrast to mammalian SQR, the membrane anchor of E. coli QFR lacks heme; thus, this prosthetic group can be excluded as a spin relaxation enhancer. The trinuclear iron-sulfur cluster FR3 in the [3Fe-4S](1+) state is suggested as the dominant spin relaxation enhancer of the SQ(FR) spins in this enzyme. E. coli QFR activity and the fast relaxing SQ species observed in the mutant enzyme are sensitive to the inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO). In wild-type E. coli QFR, HQNO causes EPR spectral line shape perturbations of the iron-sulfur cluster FR3. Similar spectral line shape changes of FR3 are caused by the FrdC E29L mutation, without addition of HQNO. This indicates that the SQ and the inhibitor-binding sites are located in close proximity to the trinuclear iron-sulfur cluster FR3. The data further suggest that this site corresponds to the proximal quinone-binding site in E. coli QFR.

Binding Sites↗
PubMed2001

Heat shock protein-chaperoned peptides but not free peptides introduced into the cytosol are presented efficiently by major histocompatibility complex I molecules.

The studies reported here bear on the events in the cytosol that lead to trafficking of peptides during antigen processing and presentation by major histocompatibility complex (MHC) I molecules. We have introduced free antigenic peptides or antigenic peptides bound to serum albumin or to cytosolic heat shock proteins hsp90 (and its endoplasmic reticular homologue gp96) or hsp70 into the cytosol of living cells and have monitored the presentation of the peptides by appropriate MHC I molecules. The experiments show that (i) free peptides or serum albumin-bound peptides, introduced into the cytosol, become ligands of MHC I molecules at a far lower efficiency than peptides chaperoned by any of the heat shock proteins tested and (ii) treatment of cells with deoxyspergualin, a drug that binds hsp70 and hsp90 with apparent specificity, abrogates the ability of cells to present antigenic peptides through MHC I molecules, and introduction of additional hsp70 into the cytosol overcomes this abrogation. These results suggest for the first time a functional role for cytosolic chaperones in antigen processing.

Amino Acid Sequence↗
PubMed1993

Identification of human cancers deficient in antigen processing.

Intracellular antigens must be processed before presentation to CD8+ T cells by major histocompatibility complex (MHC) class I molecules. Using a recombinant vaccinia virus (Vac) to transiently express the Kd molecule, we studied the antigen processing efficiency of 26 different human tumor lines. Three cell lines, all human small cell lung carcinoma, consistently failed to process endogenously synthesized proteins for presentation to Kd-restricted, Vac-specific T cells. Pulse-chase experiments showed that MHC class I molecules were not transported by these cell lines from the endoplasmic reticulum to the cell surface. This finding suggested that peptides were not available for binding to nascent MHC molecules in the endoplasmic reticulum. Northern blot analysis of these cells revealed low to nondetectable levels of mRNAs for MHC-encoded proteasome components LMP-7 and LMP-2, as well as the putative peptide transporters TAP-1 and TAP-2. Treatment of cells with interferon gamma enhanced expression of these mRNAs and reversed the observed functional and biochemical deficits. Our findings suggest that downregulation of antigen processing may be one of the strategies used by tumors to escape immune surveillance. Potential therapeutic applications of these findings include enhancing antigen processing at the level of the transcription of MHC-encoded proteasome and transporter genes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Subunit interaction maps for the regulatory particle of the 26S proteasome and the COP9 signalosome.

The 26S proteasome plays a major role in eukaryotic protein breakdown, especially for ubiquitin-tagged proteins. Substrate specificity is conferred by the regulatory particle (RP), which can dissociate into stable lid and base subcomplexes. To help define the molecular organization of the RP, we tested all possible paired interactions among subunits from Saccharomyces cerevisiae by yeast two-hybrid analysis. Within the base, a Rpt4/5/3/6 interaction cluster was evident. Within the lid, a structural cluster formed around Rpn5/11/9/8. Interactions were detected among synonymous subunits (Csn4/5/7/6) from the evolutionarily related COP9 signalosome (CSN) from Arabidopsis, implying a similar quaternary arrangement. No paired interactions were detected between lid, base or core particle subcomplexes, suggesting that stable contacts between them require prior assembly. Mutational analysis defined the ATPase, coiled-coil, PCI and MPN domains as important for RP assembly. A single residue in the vWA domain of Rpn10 is essential for amino acid analog resistance, for degrading a ubiquitin fusion degradation substrate and for stabilizing lid-base association. Comprehensive subunit interaction maps for the 26S proteasome and CSN support the ancestral relationship of these two complexes.

Arabidopsis Proteins↗
PubMed1994

Interferon-gamma induces different subunit organizations and functional diversity of proteasomes.

To obtain information on the role of proteasomes in the immune system, we examined the effect of a major immunomodulatory cytokine, gamma interferon (IFN-gamma), on the expressions, structures, and functions of proteasomes. IFN-gamma greatly increased the levels of the mRNAs encoding LMP2 and LMP7, putative immuno-proteasome subunits encoded by genes within the class II MHC region, and these two subunits synthesized were assembled completely into the proteasomal multi-subunit complex in various types of human cells. The subunit organization of proteasome changed in response to IFN-gamma stimulation, due to assembly of newly synthesized subunits through up- and down-expressions of at least 6 proteasome genes including LMP2/LMP7 without change in the structure of pre-existing proteasomes. Interestingly, IFN-gamma dramatically stimulated the trypsin-like and chymotrypsin-like activities of the multifunctional proteasome and depressed the peptidylglutamyl-peptide-hydrolyzing activity, without affecting the activity for ATP-, ubiquitin-dependent proteolysis. These results indicate that IFN-gamma modifies not only the structural organization of the proteasome, but also its functions. Based on these findings, we discuss the role in the antigen processing/presentation pathway of proteasomes with functional diversity acquired through alteration of their subunit assembly in response to IFN-gamma stimulation.

Adenosine Triphosphate↗
PubMed1999

Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation.

ATP-dependent nucleosome remodeling and core histone acetylation and deacetylation represent mechanisms to alter nucleosome structure. NuRD is a multisubunit complex containing nucleosome remodeling and histone deacetylase activities. The histone deacetylases HDAC1 and HDAC2 and the histone binding proteins RbAp48 and RbAp46 form a core complex shared between NuRD and Sin3-histone deacetylase complexes. The histone deacetylase activity of the core complex is severely compromised. A novel polypeptide highly related to the metastasis-associated protein 1, MTA2, and the methyl-CpG-binding domain-containing protein, MBD3, were found to be subunits of the NuRD complex. MTA2 modulates the enzymatic activity of the histone deacetylase core complex. MBD3 mediates the association of MTA2 with the core histone deacetylase complex. MBD3 does not directly bind methylated DNA but is highly related to MBD2, a polypeptide that binds to methylated DNA and has been reported to possess demethylase activity. MBD2 interacts with the NuRD complex and directs the complex to methylated DNA. NuRD may provide a means of gene silencing by DNA methylation.

Adenosine Triphosphatases↗
PubMed1997

Characterization of rabbit antisera elicited with human LMP2- and LMP7-specific peptides and recombinant proteins.

Anti-human LMP2 and anti-human LMP7 sera with a titer of at least 1:10,000 were developed by immunizing rabbits with LMP2- and LMP7-specific peptides corresponding to C-terminal regions of each subunit or with TrxLMP2 and TrxLMP7 recombinant proteins. IgG antibodies elicited by immunization with LMP-specific peptides or recombinant proteins displayed reactivity with their respective immunogens in ELISA. Furthermore, antibodies elicited with both types of immunogens recognize native and recombinant LMP2 and LMP7 subunits in Western blotting and are able to immunoprecipitate LMP2 and LMP7 as components of the 20S proteasome from lymphoid cell lysates. In ELISA, a subpopulation of the antibodies generated with LMP peptides and recombinant proteins corresponding to one LMP subunit is cross-reactive with the other one. This antibody subpopulation was not detectable in the affinity-purified antibody populations isolated by passing antisera over the corresponding immunogen. Neither anti-LMP2 nor anti-LMP7 sera displayed cross-reactivity with the homologous proteasome subunits Delta and MB1. In immunohistochemical reactions affinity-purified anti-LMP2 and anti-LMP7 antibodies stained cells in both frozen and formalin-fixed tissue sections of normal skin. These results indicate that the anti-LMP2 and anti-LMP7 sera elicited with peptides and recombinant proteins are both useful reagents for biochemical characterization of LMP2 and LMP7 and to analyze their expression in normal and transformed cells.

Amino Acid Sequence↗