Rapid degradation of a large fraction of newly synthesized proteins by proteasomes.

MHC class I molecules function to present peptides eight to ten residues long to the immune system. These peptides originate primarily from a cytosolic pool of proteins through the actions of proteasomes, and are transported into the endoplasmic reticulum, where they assemble with nascent class I molecules. Most peptides are generated from proteins that are apparently metabolically stable. To explain this, we previously proposed that peptides arise from proteasomal degradation of defective ribosomal products (DRiPs). DRiPs are polypeptides that never attain native structure owing to errors in translation or post-translational processes necessary for proper protein folding. Here we show, first, that DRiPs constitute upwards of 30% of newly synthesized proteins as determined in a variety of cell types; second, that at least some DRiPs represent ubiquitinated proteins; and last, that ubiquitinated DRiPs are formed from human immunodeficiency virus Gag polyprotein, a long-lived viral protein that serves as a source of antigenic peptides.

Dissecting the multifactorial causes of immunodominance in class I-restricted T cell responses to viruses.

Following influenza virus infection, the numbers of mouse TCD8+ cells responding to five different determinants vary more than 50-fold in primary responses but less so in secondary responses. Surprisingly, each determinant elicits a highly diverse and highly sensitive TCD8+ response. Inefficient antigen processing by virus-infected cells accounts for the poor immunogenicity of just one of the subdominant determinants. Overexpressing class I-peptide complexes using vaccinia virus revealed that the poor immunogenicity of two subdominant determinants reflects limitations in T cell responses unrelated to TCR diversity or sensitivity. Despite greatly enhanced expression, the immunodominant determinant is actually less immunogenic when overexpressed by vaccinia virus. Immunodominance is also modulated by determinant-specific variations in the capacity of TCD8+ to suppress responses to other determinants.

Intracellular localization of proteasomal degradation of a viral antigen.

To better understand proteasomal degradation of nuclear proteins and viral antigens we studied mutated forms of influenza virus nucleoprotein (NP) that misfold and are rapidly degraded by proteasomes. In the presence of proteasome inhibitors, mutated NP (dNP) accumulates in highly insoluble ubiquitinated and nonubiquitinated species in nuclear substructures known as promyelocytic leukemia oncogenic domains (PODs) and the microtubule organizing center (MTOC). Immunofluorescence revealed that dNP recruits proteasomes and a selective assortment of molecular chaperones to both locales, and that a similar (though less dramatic) effect is induced by proteasome inhibitors in the absence of dNP expression. Biochemical evidence is consistent with the idea that dNP is delivered to PODs/MTOC in the absence of proteasome inhibitors. Restoring proteasome activity while blocking protein synthesis results in disappearance of dNP from PODs and the MTOC and the generation of a major histocompatibility complex class I-bound peptide derived from dNP but not NP. These findings demonstrate that PODs and the MTOC serve as sites of proteasomal degradation of misfolded dNP and probably cellular proteins as well, and imply that antigenic peptides are generated at one or both of these sites.

Generating MHC class I ligands from viral gene products.

MHC class I molecules function to present peptides comprised of eight to 11 residues to CD8+ T lymphocytes. Here we review the efforts of our laboratory to understand how cells generate such peptides from viral gene products. We particularly focus on the nature of substrates acted on by cytosolic proteases, the contribution of proteasomes and non-proteasomal proteases to peptide generation, the involvement of ubiquitination in peptide generation, the intracellular localization of proteasome generation of antigenic peptides, and the trimming of peptides in the endoplasmic reticulum.

Dissociation of proteasomal degradation of biosynthesized viral proteins from generation of MHC class I-associated antigenic peptides.

To study the role of proteasomes in Ag presentation, we analyzed the effects of proteasome inhibitors Cbz-Leu-Leu-Leucinal and lactacystin on the ability of mouse fibroblast cells to present recombinant vaccinia virus gene products to MHC class I-restricted T cells. The effects of the inhibitors depended on the determinant analyzed. For influenza virus nucleoprotein (NP), presentation of the immunodominant Kk-restricted determinant (NP(50-57)) was marginally inhibited, whereas presentation of the immunodominant Kd-restricted determinant (NP(147-155)) was enhanced, particularly by lactacystin. Biochemical purification of peptides confirmed that lactacystin enhanced the generation of Kd-NP(147-155) complexes fourfold. Lactacystin also enhanced the recovery of one Kd-restricted vaccinia virus determinant from HPLC fractions, while inhibiting recovery of another. The inhibitors were used at sufficient concentrations to block presentation of biosynthesized full-length OVA and to completely stabilize a rapidly degraded chimeric ubiquitin-NP fusion protein. Strikingly, presentation of antigenic peptides from this protein was unaffected by proteasome inhibitors. We also observed that proteasome inhibitors induced expression of cytosolic and endoplasmic reticulum stress-responsive proteins. These data demonstrate first that the processes of protein degradation and generation of antigenic peptides from cytosolic proteins can be dissociated, and second that effects of proteasome inhibitors on Ag presentation may reflect secondary effects on cellular metabolism.

TAP-independent delivery of antigenic peptides to the endoplasmic reticulum: therapeutic potential and insights into TAP-dependent antigen processing.

We have taken several approaches to investigate the capacity of the secretory pathway to liberate major histocompatibility complex (MHC) class I-restricted antigenic peptides from precursor polypeptides. Cells lacking the peptide transporter (TAP) are unable to deliver peptides from cytosolic antigens to class I molecules. TAP can be bypassed by targeting peptides directly to the endoplasmic reticulum (ER) using NH2-terminal signal sequences. This results in the generation of enormous numbers of MHC class I complexes (50,000 peptides/cell), and recombinant vaccinia viruses expressing such peptides are highly immunogenic. In contrast to signal sequence-targeted peptides, peptides are liberated very inefficiently from internal locations in ER-targeted full-length proteins, indicating that the secretory pathway has a limited capacity for generating antigenic peptides from most polypeptide contexts. We have, however, identified a location in proteins from which peptides can be liberated in numerous contexts in the secretory pathway. Placing a number of different peptides at the COOH termini of a secreted protein and two proteins with type II membrane anchors resulted in their TAP-independent presentation. These findings demonstrate that the secretory compartment possesses proteases able to liberate COOH-terminal antigenic peptides from virtually any context, entirely consistent with a role for these proteases in the processing of TAP-transported antigenic peptide precursors.

CD4 glycoprotein degradation induced by human immunodeficiency virus type 1 Vpu protein requires the function of proteasomes and the ubiquitin-conjugating pathway.

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a type I anchored integral membrane phosphoprotein with two independent functions. First, it regulates virus release from a post-endoplasmic reticulum (ER) compartment by an ion channel activity mediated by its transmembrane anchor. Second, it induces the selective down regulation of host cell receptor proteins (CD4 and major histocompatibility complex class I molecules) in a process involving its phosphorylated cytoplasmic tail. In the present work, we show that the Vpu-induced proteolysis of nascent CD4 can be completely blocked by peptide aldehydes that act as competitive inhibitors of proteasome function and also by lactacystin, which blocks proteasome activity by covalently binding to the catalytic beta subunits of proteasomes. The sensitivity of Vpu-induced CD4 degradation to proteasome inhibitors paralleled the inhibition of proteasome degradation of a model ubiquitinated substrate. Characterization of CD4-associated oligosaccharides indicated that CD4 rescued from Vpu-induced degradation by proteasome inhibitors is exported from the ER to the Golgi complex. This finding suggests that retranslocation of CD4 from the ER to the cytosol may be coupled to its proteasomal degradation. CD4 degradation mediated by Vpu does not require the ER chaperone calnexin and is dependent on an intact ubiquitin-conjugating system. This was demonstrated by inhibition of CD4 degradation (i) in cells expressing a thermally inactivated form of the ubiquitin-activating enzyme E1 or (ii) following expression of a mutant form of ubiquitin (Lys48 mutated to Arg48) known to compromise ubiquitin targeting by interfering with the formation of polyubiquitin complexes. CD4 degradation was also prevented by altering the four Lys residues in its cytosolic domain to Arg, suggesting a role for ubiquitination of one or more of these residues in the process of degradation. The results clearly demonstrate a role for the cytosolic ubiquitin-proteasome pathway in the process of Vpu-induced CD4 degradation. In contrast to other viral proteins (human cytomegalovirus US2 and US11), however, whose translocation of host ER molecules into the cytosol occurs in the presence of proteasome inhibitors, Vpu-targeted CD4 remains in the ER in a transport-competent form when proteasome activity is blocked.

Introduction of a glycosylation site into a secreted protein provides evidence for an alternative antigen processing pathway: transport of precursors of major histocompatibility complex class I-restricted peptides from the endoplasmic reticulum to the cytosol.

We found that the presentation of a H-2Kd-restricted determinant from influenza virus nucleoprotein (NP) to T cells is strictly dependent on expression of the transporter associated with antigen presentation (TAP), regardless of whether NP is expressed as a cytosolic or secreted NP (SNP). Introducing an N-linked glycosylation site into the determinant selectively reduced presentation of SNP. This indicates that glycosylation does not interfere with TAP-transported peptides, and therefore that cytosolic peptides derived from SNP must have been exposed to the glycosylation machinery of the endoplasmic reticulum (ER) before their existence in the cytosol. Based on these findings, we propose that TAP-dependent processing of at least some ER-targeted proteins entails the reimportation of protein from the secretory pathway to the cytosol, where the protein is processed via the classical pathway.

The generation of MHC class I-associated peptides is only partially inhibited by proteasome inhibitors: involvement of nonproteasomal cytosolic proteases in antigen processing?

The proteasome is believed to participate in the generation of a large percentage of peptide ligands for MHC class I molecules. This conclusion is based largely on the activities of peptidyl aldehydes that block proteasome activity. We tested the ability of a panel of proteasome inhibitors to affect the generation of MHC class I binding peptides in mouse L929 cells. Included in the panel are peptidyl aldehydes and a microbial product, lactacystin, that blocks proteasome activity in a distinct and more specific manner. Contrary to expectations, proteasome inhibitors failed to block the generation of a large portion of high affinity peptides as inferred by measuring cell surface expression of newly synthesized MHC class I molecules. These findings were confirmed by examining the effects of the inhibitors on the presentation of individual antigenic determinants from endogenously synthesized or exogenously delivered influenza virus proteins. Presentation of peptides derived from exogenous basic polymerase 1, endogenous basic polymerase 1, and nonstructural-1 proteins was decreased by inhibitors in a manner consistent with proteasomal involvement. Presentation of peptides derived from endogenous nucleoprotein was not significantly affected by the proteasome inhibitors, while presentation of exogenous hemagglutinin and nucleoprotein was enhanced by the proteasome inhibitors. These data are consistent with the involvement of both proteasomes and nonproteasomal cytosolic proteases in the generation of a significant portion of MHC class I binding peptides.

MHC class I-associated peptides produced from endogenous gene products with vastly different efficiencies.

We compared the efficiency of generating antigenic peptides from various polypeptide contexts expressed by recombinant vaccinia viruses. These included full-length influenza virus nucleoprotein (NP(1-498)), two truncated forms, and cytosolic and endoplasmic reticulum-targeted minimal peptides. Two peptides were studied, NP(50-57) (Kk-restricted) and NP(147-155) (Kd-restricted). The efficiency of peptide generation was measured in cytotoxicity assays by determining 1) the kinetics of presentation following infection using brefeldin A to block additional presentation and 2) the concentration of anti-class I mAbs required to block presentation. The two determinants behaved similarly, being presented most efficiently from minigene products, with intermediate efficiency from fragments, and least efficiently from NP(1-498). Direct quantitation of HPLC-purified peptides supported the validity of these simple methods to roughly estimate the efficiency of class I Ag presentation. It also surprisingly revealed that 60- to 90-fold more NP(50-57) than NP(147-155) peptide was present in cells expressing NP(1-498) or a rapidly degraded fragment (for NP(1-498), 1800 peptides/cell of NP(50-57) vs 30 peptides/cell of NP(147-155)). By contrast, nearly identical (and much greater) amounts of peptides were recovered from cells expressing minigene products (55,000 copies of either peptide/cell). These findings demonstrate 1) that immunodominant peptides from the same protein can be generated with vastly different efficiencies, and 2) that cytosolic or endoplasmic reticulum-targeted minigene products are presented far more efficiently than longer polypeptides.

Defective ribosomal products (DRiPs): a major source of antigenic peptides for MHC class I molecules?

MHC class I molecules predominantly bind to peptides derived from a cytosolic pool of polypeptides. Little is known about the nature of the polypeptides that serve as substrates for peptidogenic cytosolic proteases. We propose that a significant source of self and viral peptides are defective ribosomal products (DRiPs), which consist of prematurely terminated polypeptides and misfolded polypeptides produced from translation of bona fide mRNAs in the proper reading frame. DRiPs are produced entropically, due to the inevitable imperfections inherent to protein synthesis or folding. To accelerate recognition of cells harboring intracellular parasites such as viruses, DRiP formation may be enhanced by changes in the cellular physiology induced by infection or by exposure of cells to cytokines released at the site of inflammation.

C3 binds with similar efficiency to Fab and Fc regions of IgG immune aggregates.

The covalent binding reaction of the third component of complement (C3) with rabbit IgG immune aggregates has been studied by enzymic digestion of C3b-IgG adducts. In these adducts C3b was radioactively labeled in the free thiol group generated during activation of the internal thioester of C3. Trypsin digestion of 14C-labeled C3b-IgG adducts degrades C3b to a small antibody-bound 14C-labeled C3 fragment (14C-C3frg), whereas the antibody remains unaltered. Papain digestion of trypsin-treated 14C-C3frg-IgG complexes generated Fc and Fab fragments bearing equivalent amounts of covalently bound 14C-C3frg (43% and 40%, of the total C3 present in the aggregates, respectively). Hydroxylamine treatment of the 14C-C3frg-Fab and 14C-C3frg-Fc complexes released a 14C-C3frg of similar size (about 3-4 kDa) in which the N-terminal residue was the radiolabeled Cys1010. A fragment with the same radioactive N terminus and characteristics was obtained by sequential trypsin and papain digestion of purified C3 labeled with iodo-[14C] acetamide. Affinity-purified 14C-C3frg-Fc complexes digested with pepsin generated a mixture of radioactive peptides, most probably complexes formed by 14C-C3frg and C gamma 2 or the hinge digestion products, and 14C-C3frg-pFc' complexes. The latter was also immunoprecipitated with anti-Fc-Sepharose from the pepsin digestion supernatants of 14C-labeled-C3b-IgG complexes. Taken together these data indicate that, during complement activation through the alternative pathway by IgG immune aggregates, C3 is not bound to a single site on the antibody molecule. Both Fab and Fc regions of IgG are equally efficient targets for C3 anchorage. In addition, the data confirm the pFc' as a region of C3 attachment within the Fc portion, and strongly suggest that C3b is bound either to the C gamma 2 domain or the hinge or both.

Arginine residues of the globular regions of human C1q involved in the interaction with immunoglobulin G.

The immunoglobulin G binding site in the globular regions of human complement subcomponent C1q has been investigated by chemical modification of histidine residues with diethylpyrocarbonate and arginine residues with phenylglyoxal and cyclohexane-1,2-dione (CHD). Only the modification of arginine residues with CHD fulfills the requirements of a specific modification without unwanted side reactions. Specific modification of arginine residues with CHD results in loss of immune complex recognition without affecting the binding of C1r2S2 to form C1. The gross structure of C1q is not changed by CHD treatment, and immune complex binding is restored to 82% of the control upon NH2OH treatment. Enzymic digestion and isolation of the modified peptides indicate that the modification by CHD of 4 to 5 arginine residues (A162, B114, B129, C156, and possibly B163) per C1q globular "head" abolishes the ability of C1q to interact with immune complexes. These residues define two areas (and possible binding sites for IgG) on the globular region of C1q: B114-B129 (site 1) and A162-(B163)-C156 (site 2). Sequence comparison and solvent exposure predictive studies favor site 2 as the immunoglobulin G binding site on the globular regions of C1q, although the participation of site 1 cannot be ruled out.

Formation of covalently linked C3-C3 dimers on IgG immune aggregates.

Upon activation of the complement system by IgG immune aggregates several components become tightly bound to the aggregates. The covalent interaction of C3 with immune complexes is essential for the solubilization and inhibition of immune precipitation of the complexes. It has recently been reported that on erythrocytes that have a fixed complement, activated C3 can become involved in the formation of C3b-C3b covalent dimers, which acts as high-affinity binding sites for C5 (Kinoshita, T., Takata, Y., Kozono, H., Takeda, J., Hong, K. and Inoue, K., J. Immunol. 1988 141: 3895). To characterize the molecular composition of immune aggregates that have fixed complement by the alternative pathway, we have investigated whether such C3b-C3b dimers are formed in IgG immune complexes. For this purpose immune aggregates bearing covalently bound C3 were analyzed by two-dimensional gel electrophoresis and the resolved bands transferred to polyvinylidene difluoride membranes and sequenced. When immune aggregates were incubated with serum for 15 min at 37 degrees C, the major high-molecular mass bands detected by gel electrophoresis corresponded to heavy chain-C3 alpha 65 and C3 alpha 65-C3 alpha 43 (derived from iC3b-iC3b-IgG) covalent complexes. If K76COONa, an inhibitor of factor I, was added to the serum, before incubation with the immune complexes, then the major C3 alpha fragment detected on the complexes corresponded to the C3 alpha' chain (105 kDa) and not C3 alpha 65. Hence C3b-C3b covalent dimers are readily formed on the immune aggregates incubated with normal human serum, and are degraded to iC3b-iC3b by factor I. The second C3b molecule was shown to be bound to the C3 alpha 43 region (C-terminal portion of the C3 alpha' chain) of the first C3b molecule, which was itself covalently bound to the heavy chain of IgG. Covalent complexes of heavy chain-(C3 alpha 65)2 molecular composition were also detected, but their precise bonding pattern has not been established.

Proteolytic activity of the different fragments of factor B on the third component of complement (C3). Involvement of the N-terminal domain of Bb in magnesium binding.

Kinetic experiments measuring the proteolytic activity of Bb and 33Kd fragment (the C-terminal domain of factor B) on C3 were performed in several conditions, in order to assess the role of factor B domains in the catalytic activity and magnesium binding. The experiments were carried out in fluid phase with 125I-C3 or C3(H2O) as substrates and in the presence of nonradioactive C3b as cofactor. The results indicate: (a) The C-terminal domain, 33Kd, possesses proteolytic activity on C3, which is Mg2(+)-independent, whereas proteolysis by Bb is enhanced in 5 mM Mg2+. (b) C3b behaves as cofactor of 33Kd proteolytic activity on C3 and factor H is able to inhibit this activity. (d) Kinetics of C3 proteolysis by 33Kd shows a lag phase which is also displayed by Bb in the absence but not in the presence of Mg2+. Taken together these data are consistent with the involvement of the N-terminal domain of Bb in Mg2+ binding, which results in an enhancement of the proteolytic activity on C3 of the adjacent C-terminal domain. A C3 convertase model accounting for these results is presented.

Separation of active and inactive forms of the third component of human complement, C3, by fast protein liquid chromatography (FPLC).

C3(H2O), an inactive form of C3 present to a variable extent in most C3 preparations, has been isolated in 40 min from previously purified C3 using FPLC ion exchange chromatography on a Mono Q column. As many as six peaks were obtained from some C3 preparations, corresponding to different molecular forms of the protein. One of these peaks consisted of a molecular form of C3 with intact alpha and beta chains, a free sulfhydryl group but no hemolytic activity and was identified as C3(H2O). C3(H2O) eluted as a homogeneous peak well resolved from native C3, C3b, high molecular weight aggregates and small degradation fragments. The same C3(H2O) peak was generated from native C3 by repeated freeze-thaw cycles or NH2OH treatment. C3(H2O) alpha chain appeared as a doublet about 2 kDa heavier than native C3 alpha chain in low cross-linked gels. Two forms of C3b could be separated on the Mono S column, both able to form the C3 convertase. The present report describes a very fast method to resolve and isolate to homogeneity C3(H2O) and native C3 from C3 preparations. Both molecular forms of C3 are very suitable for studies of the initial and amplification C3 convertases of the alternative pathway of complement.

C3 binds covalently to the C gamma 3 domain of IgG immune aggregates during complement activation by the alternative pathway.

Ovalbumin-antiovalbumin IgG immune aggregates were incubated with normal human serum in the presence of iodo[1-14C]acetamide, in conditions in which only the alternative pathway of complement was activated. The [14C]C3b-IgG covalent complexes formed were digested with pepsin, and analysed by SDS/polyacrylamide-gel electrophoresis and fluorography. Covalent complexes of [14C]C3-Fd and [14C]C3-pFc' were visualized, demonstrating that, during complement activation by the alternative pathway, C3 is covalently incorporated into the C gamma 3 domain of IgG, as well as into the Fd region. The C gamma 2 domain becomes protected from pepsin action by the bound C3b. All the covalent linkages between C3 and the IgG were sensitive to hydroxylamine. When [14C]C3-pFc' covalent complexes were treated with 1 M-NH2OH and loaded onto a Bio-Gel P-4 column, a radioactive peak of 3 kDa was obtained. The material released from [14C]C3-pFc' and [14C]C3-F(ab')2 complexes after treatment with 1 M-NH2OH was mixed and analysed in the Bio-Gel P-4 column. A similar radioactive peak of 3 kDa was obtained. When this peak, either from [14C]C3-pFc' alone or from the mixture of [14C]C3-F(ab')2 and [14C]C3-pFc', was fractionated by h.p.l.c., virtually the same radioactive peptide profile was obtained, indicating that very similar C3 peptides remained covalently bound to both regions (Fab and C gamma 3) of the antibody molecule. It is suggested that C3 bound to the C gamma 3 domain of IgG may interfere with the Fc-Fc interactions of immune aggregates and thus may be involved in several biological properties displayed by these complement-activating aggregates.

Formation of covalent complexes between the fourth component of human complement and IgG immune aggregates.

The binding properties of activated C4 to immune complexes (ovalbumin-rabbit IgG antiovalbumin) were studied by using 125I-IgG in the immune complexes or performing the C4 binding assays in the presence of 14C-iodoacetamide. High molecular weight complexes formed between C4 and IgG could be detected by the incorporation of 14C-iodoacetamide in the -SH group generated in the nascent C4b during the activation process. The same complexes with an apparent molecular weight of 180,000 daltons were detected when the immune aggregates contained 125I-IgG. Two-dimensional SDS-PAGE analysis of the C4b-IgG covalent complexes indicated: In the absence of control proteins, the complexes are formed by the alpha'-chain of C4b and the H chain of the antibody. The alpha'-H complexes are 36% sensitive to hydroxylamine and 64% resistant. This is consistent with the presence of two populations of C4, which are not equivalent in their covalent binding with immune complexes. Covalent complexes C4-C4b or C4b(like)-C4b(like) are generated during the C4 activation and they are detected as alpha-alpha' or alpha-alpha complexes, respectively. Interaction of C4b with the L chain of the antibody molecule also seems to occur, but to a lesser extent than with the H chain.

The interaction of 1-anilino-8-naphthalene sulphonate with human C1q.

C1q has 12 binding sites for 1-anilino-8-naphthalene sulphonate (ANS), two per peripheral subunit. This number increases to 18 upon weak-acid-induced conformational transition in the globular heads. One ANS binding site is present in each C gamma 2 domain of human IgG. ANS is bound by C1q with a higher affinity (Ka = 2.07 X 10(6) M-1) than by the Fc fragment (Ka = 9.07 X 10(4) M-1) of human IgGl. Hence the inhibitory capacity of C1q binding to IgG immune complexes of ANS probably reflects its preferential binding to the globular heads of C1q. The characteristics of ANS-C1q binding may in part explain the hydrophobic component of the C1q-IgG interaction. It is suggested that an ionic-hydrophobic two-step process is involved in the contact between C1q and IgG.