![Inhibition by Peptide Aldehyde Is Reversible (A) Reversible inhibition of ovalbumm presentation. One group of LB27.4 cells was premcubated with (open triangles) or without LLnL (closed circles) (50 gM) for 1 hr, electroporated with ovalbumin (30 mg/ml) with or without LLnL (50 gM), incubated in the continued presence or absence of LLnL (50 p.M) for an additional 1 hr, and then fixed. Another group of LB27.4 cells (open triangles) was exposed to LLnL (50 p.M) under the same conditions and for the same length of time (2 hr), but the inhibitor was subsequently removed by washing. Subsequently, ovalbumin was introduced into these later cells by electroporat~on, and they were incubated for an additional 1 hr without LLnL and then fixed. Antigen presentation was assayed as described in Figure 4. In the absence of antigen, background responses were <1,400 cpm. (B) Reversible inhibition of MHC class I assembly. Autoradiograms of immunoprecip~tated K b heterodimers from RMA cells treated without (contrN) or wrth LLnL (100 i.tM) and then [3SS]methionine labeled and solubilized immediately (as described in [A]) or washed and allowed to recover for 2 hr at 37°C before metabolic labeling and detergent lysls. Detergent lysates were precleared and immunoprecipitated with Y3 monoclonal antibody (anti-K b heterodimers) and analyzed on SDS-PAGE gels. The positions of the class I heavy chains and I~a-microglobulin on the autoradiograms are indicated by H and [32, respectively](https://www.researchgate.net/profile/Ross-Stein/publication/287891176/figure/fig1/AS:812571254857730@1570743492381/Inhibition-by-Peptide-Aldehyde-Is-Reversible-A-Reversible-inhibition-of-ovalbumm_Q320.jpg)
![LLnL and MGl15 Inhibit the Assembly of K" Class I Molecules Autoradiograms of immunoprecipitated K b heterodimers (H plus ~2), free heavy chains (H), or free light chains (~2) from [~S]methioninelabeled liMA cells treated with (plus) or without (minus) inhibitors. (A), LLnL-treated cells; (B), LLnL, LLM, or E644reated cells; (C), MG115-treated cells, lIMA cells (15 x 106) were treated with or without peptlde aldheydes (100 I~M, unless indicated otherwise) or E64 (25 ~M) for 2.5 hr with [sSS]methionine (0.7 mCi/ml) added for the last 30 min of incubation. Detergent lysates were precleared and immunoprecipitated sequentially with Y3 (anti-K b H plus 62 chain), rabbit anti-exon 8 (anti-K b H chain), B22.249 (anti-D e H plus 132 chains), 28.14.8S (anti-D b H chain), and $19.8.503 (anti-132) antibodies and analyzed on SDS-PAGE gels. The positions of the class I heavy chains and 13~-microglobulin on the autoradiograrns are indicated by H and 1~2, respectively.](https://www.researchgate.net/profile/Ross-Stein/publication/287891176/figure/fig2/AS:812571254853632@1570743492502/LLnL-and-MGl15-Inhibit-the-Assembly-of-K-Class-I-Molecules-Autoradiograms-of_Q320.jpg)
![Exogenous Peptides Reconstitute Class I Assembly in LLnL-Treated Cell Lysates Autoradiogram of immunoprecipitated class I heterodimers from detergent lysates of [~S]methionine-labeled RMA cells treated with (plus) or without (minus) LLnL (100 p.M) to which SIINFEKL and ASNENMETM peptides (plus) or no peptides (minus) were added. Treatment with inhibutors, and immunoprecipitations with Y3 (anti-K b heterodimers) (A) and B22.249 (anti-D b heterodimers) (B), were performed and analyzed as described in Figure 6. Peptides were present in the lysates during the preclearing steps (approximately 20 hr) before the specific immunoprecipitations were performed. The positions of the class I heavy chains and l~2-microglobulin on the autoradiograms are indicated by H and I]2, respectively.](https://www.researchgate.net/profile/Ross-Stein/publication/287891176/figure/fig3/AS:812571254853634@1570743492816/Exogenous-Peptides-Reconstitute-Class-I-Assembly-in-LLnL-Treated-Cell-Lysates_Q320.jpg)
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Inhibitors of the proteasome block the degradation of most cell proteins and the generetaion of peptides presented on MHC class
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... Proteasomes catalyze the majority of protein degradation in all mammalian cells (Rock et al., 1994). Regardless of the composition of a proteasome, as a result of the proteasome-dependent degradation, proteins are cleaved into small peptides that range in size from 3-25 amino acids (Kisselev et al., 1999;Yewdell et al., 2003). ...
... Regardless of the composition of a proteasome, as a result of the proteasome-dependent degradation, proteins are cleaved into small peptides that range in size from 3-25 amino acids (Kisselev et al., 1999;Yewdell et al., 2003). While around 97% of proteasome-derived peptides are eventually recycled into amino acids, ∼3% of the peptides have been shown to play critical roles in human biology, especially in the case of Major Histocompatibility Complex class I (MHC-I) antigen presentation in the immune system (Goldberg and Rock, 1992;Rock et al., 1994;McCarthy and Weinberg, 2015). Antigen presentation by MHC-I molecules on the surface of antigen-presenting cells is essential for cytotoxic (CD8+) T-cell activation through the interaction of the MHC-bound antigen and the T-cell receptor (TCR) to trigger an immune response against cancer, infection, or self, in the case of autoimmunity. ...
The neuronal membrane proteasome (NMP) degrades intracellular proteins into peptides that are released directly into the extracellular space, whereby they stimulate neurons to promote signaling mechanisms that remain unknown. Here, we demonstrate that neuronal stimulation promotes NMP activity and, subsequently, enhanced production of NMP peptides. We show that these neuronal activity-dependent NMP peptides can rapidly promote N-methyl-D-aspartate receptor (NMDAR)-dependent calcium influx in neurons. This leads to sustained phosphorylation of the well-defined stimulus-induced transcription factor, cyclic AMP response element (CRE)-binding protein (CREB). Downstream of these events, we identified changes to neuronal target genes which included increased expression of immediate early genes (e.g., Fos, Npas4, Egr4) and other genes known to have critical neuroregulatory roles. Further observations led to the discovery that NMP peptide-induced changes in gene expression is dependent on NMDARs and independent of AMPA receptors or voltage-gated sodium channels. These data demonstrate that NMP peptides are endogenous and selective activators of NMDA receptors and act as sufficient and novel stimuli within the context of neuronal activity-dependent signaling. This novel pathway is parallel to classic neuronal activity-dependent programs and points to NMP and its resulting peptides as potential modulators of neuronal function.
[Media: see text] [Media: see text] [Media: see text]
... 87,88 In our functional association cohort, we also found type 1 IFN-induced immunoproteasome subunits (PSMB8 and PSMB9) essential for antigen presentation and CD8 T cell activation to be upregulated in AD cases ( Figure S8). [89][90][91] Since AD is a heterogenous disease that consists of multiple endophenotypes and a "one-size-fits-all" approach is insufficient to maximize treatment efficacy, 92-94 this makes the type 1 IFN signaling pathway an attractive target to achieve a more effective and personalized treatment for AD patients with a dysregulated type 1 IFN signaling profile. ...
Background
Atopic dermatitis (AD) is a complex inflammatory disease with a strong genetic component. A singular approach of genome wide association studies (GWAS) can identify AD-associated genetic variants, but is unable to explain their functional relevance in AD. This study aims to characterize AD-associated genetic variants and elucidate the mechanisms leading to AD through a multi-omics approach.
Methods
GWAS identified an association between genetic variants at 6p21.32 locus and AD. Genotypes of 6p21.32 locus variants were evaluated against LOC100294145 expression in peripheral blood mononuclear cells (PBMCs). Their influence on LOC100294145 promoter activity was measured in vitro via a dual-luciferase assay. The function of LOC100294145 was then elucidated through a combination of co-expression analyses and gene enrichment with g:Profiler. Mendelian randomization was further used to assess the causal regulatory effect of LOC100294145 on its co-expressed genes.
Results
Minor alleles of rs116160149 and rs115388857 at 6p21.32 locus were associated with increased AD risk (p = 2.175 × 10⁻⁸, OR = 1.552; p = 2.805 × 10⁻⁹, OR = 1.55) and higher LOC100294145 expression in PBMCs (adjusted p = 0.182; 8.267 × 10⁻¹²). LOC100294145 expression was also found to be increased in those with AD (adjusted p = 3.653 × 10⁻²). The genotype effect of 6p21.32 locus on LOC100294145 promoter activity was further validated in vitro. Co-expression analyses predicted LOC100294145 protein's involvement in interleukin-27 and type 1 interferon signaling, which was further substantiated through mendelian randomization.
Conclusion
Genetic variants at 6p21.32 locus increase AD susceptibility through raising LOC100294145 expression. A multi-omics approach enabled the deduction of its pathogenesis model comprising dysregulation of hub genes involved in type 1 interferon and interleukin 27 signaling.
... Regardless of isoform, the collective action of the 20S proteasomes' multiple, catalytically diverse active sites has the potential to extensively degrade the varied complement of cellular proteins to amino acids and short peptides, a feature consistent with the proteasome's role as the predominant system for intracellular protein degradation (10,11). Several structural properties of 20S proteasomes, however, normally restrict this proteolytic function. ...
PI31 (Proteasome Inhibitor of 31,000 Daltons) is a 20S proteasome binding protein originally identified as an in vitro inhibitor of 20S proteasome proteolytic activity. Recently reported cryo-electron microscopy structures of 20S-PI31 complexes reveal a surprising structural basis for proteasome inhibition. The natively disordered proline-rich C-terminus of PI31 enters the central chamber in the interior of the cylindrical 20S proteasome and interacts directly with the proteasomes multiple catalytic threonine residues a manner predicted to inhibit their enzymatic function while evading its own proteolysis. Higher eukaryotes express an alternative form of 20S proteasome featuring genetically and functionally distinct catalytic subunits. This proteasome is expressed in tissues involved in immune function or in response to certain cytokines such as interferon-gamma and has been termed immuno-proteasome. We examined the relative effects of PI31 on constitutive and immuno- 20S proteasomes and show that PI31 inhibits the immuno-20S proteasome (20Si) to a significantly lesser degree than it inhibits constitutive 20S proteasome (20Sc). Unlike 20Sc, 20Si hydrolyzes the carboxyl-terminus of PI31and this effect contributes to the reduced inhibitory activity of PI31 towards 20Si. These results demonstrate unexpected differential interactions of PI31 with 20Sc and 20Si and document their functional consequences.
... The ubiquitin-proteasome system (UPS) is involved in multiple fundamental cellular processes through regulating more than 80% cellular proteins' degradation and function; and dysregulation of UPS has been implicated in the pathogenesis of many human diseases such as neurodegenerative disorders, viral diseases and cancer [1,2]. The process of ubiquitination is highly dynamic and is reversed by the deubiquitinases (DUBs). ...
Deubiquitinases (DUBs) play important roles in various human cancers and targeting DUBs is considered as a novel anticancer therapeutic strategy. Overexpression of ubiquitin specific protease 7 and 22 (USP7 and USP22) are associated with malignancy, therapy resistance, and poor prognosis in many cancers. Although both DUBs are involved in the regulation of similar genes and signaling pathways, such as histone H2B monoubiquitination (H2Bub1), c-Myc, FOXP3, and p53, the interdependence of USP22 and USP7 expression has never been described. In the study, we found that targeting USP7 via either siRNA-mediated knockdown or pharmaceutical inhibitors dramatically upregulates USP22 in cancer cells. Mechanistically, the elevated USP22 occurs through a transcriptional pathway, possibly due to desuppression of the transcriptional activity of SP1 via promoting its degradation upon USP7 inhibition. Importantly, increased USP22 expression leads to significant activation of downstream signal pathways including H2Bub1
and c-Myc, which may potentially enhance cancer malignancy and counteract the anticancer efficacy of USP7 inhibition. Importantly, targeting USP7 further suppresses the in vitro proliferation of USP22-knockout (USP22-Ko)
A549 and H1299 lung cancer cells and induces a stronger activation of p53 tumor suppressor signaling pathway. In addition, USP22-Ko cancer cells are more sensitive to a combination of cisplatin and USP7 inhibitor. USP7 inhibitor treatment further suppresses in vivo angiogenesis and tumor growth and induced more apoptosis in USP22-Ko cancer xenografts. Taken together, our findings demonstrate that USP7 inhibition can dramatically upregulate USP22 in cancer cells; and targeting USP7 and USP22 may represent a more effective approach for targeted cancer therapy,
which warrants further study.
Keywords Deubiquitinase, USP7, USP22, SP1, c-Myc, p53, Targeted anticancer therapy
... The ubiquitin-proteasome system (UPS) is involved in multiple fundamental cellular processes through regulating more than 80% cellular proteins' degradation and function; and dysregulation of UPS has been implicated in the pathogenesis of many human diseases such as neurodegenerative disorders, viral diseases and cancer [1,2]. The process of ubiquitination is highly dynamic and is reversed by the deubiquitinases (DUBs). ...
Deubiquitinases (DUBs) play important roles in various human cancers and targeting DUBs is considered as a novel anticancer therapeutic strategy. Overexpression of ubiquitin specific protease 7 and 22 (USP7 and USP22) are associated with malignancy, therapy resistance, and poor prognosis in many cancers. Although both DUBs are involved in the regulation of similar genes and signaling pathways, such as histone H2B monoubiquitination (H2Bub1), c-Myc, FOXP3, and p53, the interdependence of USP22 and USP7 expression has never been described. In the study, we found that targeting USP7 via either siRNA-mediated knockdown or pharmaceutical inhibitors dramatically upregulates USP22 in cancer cells. Mechanistically, the elevated USP22 occurs through a transcriptional pathway, possibly due to desuppression of the transcriptional activity of SP1 via promoting its degradation upon USP7 inhibition. Importantly, increased USP22 expression leads to significant activation of downstream signal pathways including H2Bub1 and c-Myc, which may potentially enhance cancer malignancy and counteract the anticancer efficacy of USP7 inhibition. Importantly, targeting USP7 further suppresses the in vitro proliferation of USP22-knockout (USP22-Ko) A549 and H1299 lung cancer cells and induces a stronger activation of p53 tumor suppressor signaling pathway. In addition, USP22-Ko cancer cells are more sensitive to a combination of cisplatin and USP7 inhibitor. USP7 inhibitor treatment further suppresses in vivo angiogenesis and tumor growth and induced more apoptosis in USP22-Ko cancer xenografts. Taken together, our findings demonstrate that USP7 inhibition can dramatically upregulate USP22 in cancer cells; and targeting USP7 and USP22 may represent a more effective approach for targeted cancer therapy, which warrants further study.
... Protein degradation is one of the essential mechanisms regulating levels of cellular proteins involved in crucial cellular processes such as cell cycle, development and growth, transcription, cell signaling, and antigen processing (DeMartino and Slaughter 1999). The UPS is responsible for the detoxification and targeting of damaged proteins for degradation (Coux et al. 1996;Rock et al. 1994;Jang and Chung 2022;Licchesi et al 2020;Di Dominico and Lanzillotta 2022), except membrane and extracellular proteins, which after endocytosis are degraded within the lysosomes (Sherman and Goldberg 2001). Proteins to be degraded are first marked by covalent attachment of a polyubiquitin chain to a lysine residue on the substrate and then are degraded by a large proteolytic complex, the 26S proteasome. ...
Our and other’s laboratory microarray-derived transcriptomic studies in human PD substantia nigra pars compacta (SNpc) samples have opened an avenue to concentrate on potential gene intersections or cross-talks along the dopaminergic (DAergic) neurodegenerative cascade in sporadic PD (SPD). One emerging gene candidate identified was SKP1A (p19, S-phase kinase-associated protein 1A), found significantly decreased in the SNpc as confirmed later at the protein level. SKP1 is part of the Skp1, Cullin 1, F-box protein (SCF) complex, the largest known class of sophisticated ubiquitin–proteasome/E3-ligases and was found to directly interact with FBXO7, a gene defective in PARK15-linked PD. This finding has led us to the hypothesis that a targeted site-specific reduction of Skp1 levels in DAergic neuronal cell culture and animal systems may result in a progressive loss of DAergic neurons and hopefully recreate motor disabilities in animals. The second premise considers the possibility that both intrinsic and extrinsic factors (e.g., manipulation of selected genes and mitochondria impairing toxins), alleged to play central roles in DAergic neurodegeneration in PD, may act in concert as modifiers of Skp1 deficiency-induced phenotype alterations (‘dual-hit’ hypothesis of neurodegeneration). To examine a possible role of Skp1 in DAergic phenotype, we have initially knocked down the expression of SKP1A gene in an embryonic mouse SN-derived cell line (SN4741) with short hairpin RNA (shRNA) lentiviruses (LVs). The deficiency of SKP1A closely recapitulated cardinal features of the DAergic pathology of human PD, such as decreased expression of DAergic phenotypic markers and cell cycle aberrations. Furthermore, the knocked down cells displayed a lethal phenotype when induced to differentiate exhibiting proteinaceous round inclusion structures, which were almost identical in composition to human Lewy bodies, a hallmark of PD. These findings support a role for Skp1 in neuronal phenotype, survival, and differentiation. The identification of Skp1 as a key player in DAergic neuron function suggested that a targeted site-specific reduction of Skp1 levels in mice SNpc may result in a progressive loss of DAergic neurons and terminal projections in the striatum. The injected LV SKP1shRNA to mouse SN resulted in decreased expression of Skp1 protein levels within DAergic neurons and loss of tyrosine hydroxylase immunoreactivity (TH-IR) in both SNpc and striatum that was accompanied by time-dependent motor disabilities. The reduction of the vertical movements, that is rearing, may be reminiscent of the early occurrence of hypokinesia and axial, postural instability in PD. According to the ‘dual-hit’ hypothesis of neurodegenerative diseases, it is predicted that gene–gene and/or gene–environmental factors would act in concert or sequentially to propagate the pathological process of PD. Our findings are compatible with this conjecture showing that the genetic vulnerability caused by knock down of SKP1A renders DAergic SN4741 cells especially sensitive to genetic reduction of Aldh1 and exposure to the external stressors MPP⁺ and DA, which have been implicated in PD pathology. Future consideration should be given in manipulation SKP1A expression as therapeutic window, via its induction genetically or pharmacological, to prevent degeneration of the nigra striatal dopamine neurons, since UPS is defective.
... After 2 h of incubation, APCs were fixed and cocultured with the S8L-specific T cell hybridoma cell line B3Z (33), using T cell activation as a measure for cross-presentation efficiency. These experiments revealed that cross-presentation of MS-OVA and OVA-SLP by BMC2 macrophages was sensitive to the reversible proteasome inhibitor MG-132 (40,41) and the secretory pathway inhibitor BFA (36,42) (Fig. 1C), with the interesting observation that cross-presentation of OVA-SLP was preferentially inhibited by interfering with proteasomal activity. In contrast, treatment with leupeptin, an inhibitor of cysteine, serine, and threonine proteases important for endosomal processing (36,4345), did not affect the cross-presentation efficiency detected for both Ags tested. ...
The induction of CTL responses by vaccines is important to combat infectious diseases and cancer. Biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres and synthetic long peptides are efficiently internalized by professional APCs and prime CTL responses after cross-presentation of Ags on MHC class I molecules. Specifically, they mainly use the cytosolic pathway of cross-presentation that requires endosomal escape, proteasomal processing, and subsequent MHC class I loading of Ags in the endoplasmic reticulum (ER) and/or the endosome. The vesicle SNARE protein Sec22b has been described as important for this pathway by mediating vesical trafficking for the delivery of ER-derived proteins to the endosome. As this function has also been challenged, we investigated the role of Sec22b in cross-presentation of the PLGA microsphere-encapsulated model Ag OVA and a related synthetic long peptide. Using CRISPR/Cas9-mediated genome editing, we generated Sec22b knockouts in two murine C57BL/6-derived APC lines and found no evidence for an essential role of Sec22b. Although pending experimental evidence, the target SNARE protein syntaxin 4 (Stx4) has been suggested to promote cross-presentation by interacting with Sec22b for the fusion of ER-derived vesicles with the endosome. In the current study, we show that, similar to Sec22b, Stx4 knockout in murine APCs had very limited effects on cross-presentation under the conditions tested. This study contributes to characterizing cross-presentation of two promising Ag delivery systems and adds to the discussion about the role of Sec22b/Stx4 in related pathways. Our data point toward SNARE protein redundancy in the cytosolic pathway of cross-presentation.
... Proteasomes are fundamental to the ATP-dependent proteolytic pathway in eukaryotic cells (285). In fact, most cellular protein degradation occurs through proteasome catalyzed degradation (286). The FDA approved the first proteasome inhibitor, bortezomib (PS-341), for multiple myeloma in 2003 (287, 288). ...
Cutaneous T cell lymphomas are a rare subset of non-Hodgkin’s lymphomas with predilection for the skin with immunosuppressive effects that drive morbidity and mortality. We are now appreciating that suppression of the immune system is an important step in the progression of disease. It should come as no surprise that therapies historically and currently being used to treat these cancers have immune modulating functions that impact disease outcomes. By understanding the immune effects of our therapies, we may better develop new agents that target the immune system and improve combinatorial treatment strategies to limit morbidity and mortality of these cancers. The immune modulating effect of therapeutic drugs in use and under development for cutaneous T cell lymphomas will be reviewed.
... The proteasome serves essential functions in proteostasis by degrading proteins and peptides within a cell. 30 Although β 5 26S proteolytic activity was lower in old muscle than adult muscle during WB, it was higher in old GA muscle than adult GA muscle after HU. A previous study did not identify the same increase in proteasomal activity, but the response to RE was like ours. 5 This difference in proteasomal response to disuse may arise from the difference in muscles tested between studies. ...
Background:
Skeletal muscle mass and strength diminish during periods of disuse but recover upon return to weight bearing in healthy adults but are incomplete in old muscle. Efforts to improve muscle recovery in older individuals commonly aim at increasing myofibrillar protein synthesis via mammalian target of rapamycin (mTOR) stimulation despite evidence demonstrating that old muscle has chronically elevated levels of mammalian target of rapamycin complex 1 (mTORC1) activity. We hypothesized that protein synthesis is higher in old muscle than adult muscle, which contributes to a proteostatic stress that impairs recovery.
Methods:
We unloaded hindlimbs of adult (10-month) and old (28-month) F344BN rats for 14 days to induce atrophy, followed by reloading up to 60 days with deuterium oxide (D2 O) labelling to study muscle regrowth and proteostasis.
Results:
We found that old muscle has limited recovery of muscle mass during reloading despite having higher translational capacity and myofibrillar protein synthesis (0.029 k/day ± 0.002 vs. 0.039 k/day ± 0.002, P < 0.0001) than adult muscle. We showed that collagen protein synthesis was not different (0.005 k (1/day) ± 0.0005 vs. 0.004 k (1/day) ± 0.0005, P = 0.15) in old compared to adult, but old muscle had higher collagen concentration (4.5 μg/mg ± 1.2 vs. 9.8 μg/mg ± 0.96, P < 0.01), implying that collagen breakdown was slower in old muscle than adult muscle. This finding was supported by old muscle having more insoluble collagen (4.0 ± 1.1 vs. 9.2 ± 0.9, P < 0.01) and an accumulation of advanced glycation end products (1.0 ± 0.06 vs. 1.5 ± 0.08, P < 0.001) than adult muscle during reloading. Limited recovery of muscle mass during reloading is in part due to higher protein degradation (0.017 1/t ± 0.002 vs. 0.028 1/t ± 0.004, P < 0.05) and/or compromised proteostasis as evidenced by accumulation of ubiquitinated insoluble proteins (1.02 ± 0.06 vs. 1.22 ± 0.06, P < 0.05). Last, we showed that synthesis of individual proteins related to protein folding/refolding, protein degradation and neural-related biological processes was higher in old muscle during reloading than adult muscle.
Conclusions:
Our data suggest that the failure of old muscle to recover after disuse is not due to limitations in the ability to synthesize myofibrillar proteins but because of other impaired proteostatic mechanisms (e.g., protein folding and degradation). These data provide novel information on individual proteins that accumulate in protein aggregates after disuse and certain biological processes such as protein folding and degradation that likely play a role in impaired recovery. Therefore, interventions to enhance regrowth of old muscle after disuse should be directed towards the identified impaired proteostatic mechanisms and not aimed at increasing protein synthesis.
Seven peptidyl proteasome inhibitors were tested for in vitro activity against Trypanosoma brucei bloodstream forms. Two compounds showed activity in the low nanomolar range. In general, trypanosomes were more susceptible to the compounds than were human HL-60 cells. The data support the potential of proteasome inhibitors for rational antitrypanosomal drug development.
The interaction of nominal Ag with major histocompatibility complex (MHC)-restricted T cells and accessory cells was studied by analyzing the effect of structurally related antigens on the response of antigen-specific MHC-restricted T cell hybridomas. The copolymer L-glutamic acid50-L-tyrosine50 (GT) completely inhibits the response of L-glutamic acid60-L-alanine40-L-tyrosine10 (GAT)-specific, I-Ad-restricted T cell hybridomas to GAT plus accessory cells. This inhibition is specific, as hybridomas of other specificities are not inhibited under identical conditions, and is unique to the GT antigen, as other similar copolymers are not inhibitory. The inhibitory effect is reversible by adding increasing amounts of GAT. Antigen-pulsing experiments localized the inhibition to the level of antigen-presenting cell (APC). GT-prepulsed APC are not inhibitory in cell-mixing experiments and can present other antigens. GT only inhibits the nominal antigen-directed component of a GAT-specific, autoreactive hybrid's response. Together these findings suggest that GT causes inhibition by competing for GAT association at the accessory cell. GT interferes with GAT presentation by an I-Adxb F1 APC to a BALB/c, I-Ad-restricted, but not B10, I-Ab-restricted, T cell hybridoma, and GT inhibits presentation by GAT-prepulsed APC. The implications of these findings for MHC-restricted presentation of antigen are discussed.
Antigen processing provides major histocompatibility complex (MHC) class I molecules with short peptides, which they selectively bind and present to cytotoxic T lymphocytes. The proteolytic system generating these peptides in the cytosol is unidentified, but their delivery into the endoplasmic reticulum is mediated by the TAP1-TAP2 transporter encoded in the MHC class II region. Closely linked to TAP1 and TAP2 are genes for the LMP2 and LMP7 proteins, which resemble components of proteasomes, proteolytic complexes known to degrade cytosolic proteins. This association has led to the common assumption that proteasomes function in this immunological pathway (discussed in ref. 15). We now show that the expression of stably assembled class I molecules and apparently normal peptide processing can be completely restored in the absence of LMP2 and LMP7 in the human lymphoblastoid cell line mutant 721.174 (refs 16, 17). The identity of LMP7 is directly confirmed by reconstitution of a proteasomal subunit after gene transfer. These results therefore dispute the hypothetical involvement of proteasomes in antigen processing, although a more subtle effect of LMP2 and LMP7 cannot be ruled out.
The cell hybridization technique was used for the production of 12 monoclonal antibodies against H-2Kk, H-2Db, I-Ak and I-Ek antigens. The strain distribution pattern indicated that three antibodies reacted with new H-2 and Ia determinants, respectively, while the majority of determinants defined by the monoclonal antibodies showed good correlation with H-2 and Ia determinants described by conventional alloantisera.
Monoclonal Ia antibodies showed strong reactivity with about 90% of surface IgM positive B cells, but not with T cells. In double fluorescence studies, both I-A and I-E determinants were always found to be coexpressed on the same B cells. When the high sensitivity of the fluorescence activated cell sorter was utilized, about 30 to 40% of purified lymph node T cells were found to carry both I-A and I-E antigens, although in a much lower density than B cells. In conclusion, monoclonal Ia antibodies appear to display the same serological and cellular reactivity pattern as do conventional antisera.
Two new members of the ABC superfamily of transporter genes have recently been identified within the Major Histocompatibility Complex in man, rat and mouse. Although the exact function of these genes is not known, they have been shown to be necessary for the presentation of peptides derived from the degradation of cytoplasmic protein antigens to the cellular immune systems. For this reason they have been named TAP1 and TAP2 (for Transporter associated with Antigen Presentation). Each gene encodes one membrane spanning domain and one region homologous to the ATP binding domains that characterise the superfamily. The two proteins encoded by the TAP genes from a complex that is localised to the membranes of the endoplasmic reticulum and cis-golgi. Their most likely functions is to transport short peptides, that lack signal sequences, from the cytoplasm to the endoplasmic reticulum, although the evidence for this is still indirect.
A major eukaryotic proteolytic system is known to require the covalent attachment of ubiquitin to substrates prior to their degradation, yet the proteinase involved remains poorly defined. The proteasome, a large conserved multi-subunit protein complex of the cytosol and the nucleus, has been implicated in a variety of cellular functions. It is shown here that a yeast mutant with a defective proteasome fails to degrade proteins which are subject to ubiquitin-dependent proteolysis in wild-type cells. Thus, the proteasome is part of the ubiquitin system and mediates the degradation of ubiquitin-protein conjugates in vivo.
Cells of higher vertebrates have evolved mechanisms that allow a sample of their intracellular contents to be available for surveillance by the immune system. This display of intracellular material is in the form of peptides bound to cell surface major histocompatibility complex (MHC) class I molecules. In this review, John Monaco presents a model of the mechanisms by which this takes place, based on the recent identification of a number of new genes in the MHC.
When rats are fed a protein deficient (PD) diet for 7 days, rates of proteolysis in skeletal muscle decrease by 40-50% (N. E. Tawa, Jr., and A. L. Goldberg. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E317-325, 1992). To identify the underlying biochemical adaptations, we measured different proteolytic processes in incubated muscles. The capacity for intralysosomal proteolysis, as shown by sensitivity to methylamine or lysosomal protease inhibitors, fell 55-75% in muscles from PD rats. Furthermore, extracts of muscles of PD rats showed 30-70% lower activity of many lysosomal proteases, including cathepsins B, H, and C, and carboxypeptidases A and C, as well as other lysosomal hydrolases. The fall in cathepsin B and proteolysis was evident by 3 days on the PD diet, and both returned to control levels 3 days after refeeding of the normal diet. In muscles maintained under optimal conditions, 80-90% of protein breakdown occurs by nonlysosomal pathways. In muscles of PD rats, this ATP-dependent process was also 40-60% slower. Even though overall proteolysis decreased in muscles of PD rats, their capacity for Ca(2+)-dependent proteolysis increased (by 66%), as did the activity of the calpains (+150-250%). Thus the lysosomal and the ATP-dependent processes decrease coordinately and contribute to the fall in muscle proteolysis in PD animals.
Mutagen treatment of mouse P815 tumor cells produces immunogenic mutants that express new transplantation antigens (tum- antigens) recognized by cytolytic T cells. The gene encoding tum- antigen P91A comprises 12 exons and a mutation located in exon 4 is responsible for the production of a new antigenic peptide. Transfection experiments showed that the expression of the antigen could be transferred not only by the entire gene but also by gene segments comprising only the mutated exon and parts of the surrounding introns. This was observed with subgenic regions that were not cloned in expression vectors. Antigen expression did not require the integration of the transfected gene segment into a resident P91A gene by homologous recombination. It also occurred when the subgenic segment was transfected without the usual selective gene, which comprises an eucaryotic promoter, and also without plasmid sequences, which are known to contain weak promoters. When a stop codon was introduced at the beginning of exon 4, the expression of the antigen was maintained and evidence was obtained that an ATG codon located in this region served as initiation site for the translation of the antigenic peptide. But we have not obtained evidence indicating that antigenic peptides are direct translation products rather than degradation products of entire proteins.