Crystal structure of MHC class II-associated p41 Ii fragment bound to cathepsin L reveals the structural basis for differentiation between cathepsins L and S.
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The lysosomal cysteine proteases cathepsins S and L play crucial roles in the degradation of the invariant chain during maturation of MHC class II molecules and antigen processing. The p41 form of the invariant chain includes a fragment which specifically inhibits cathepsin L but not S. The crystal structure of the p41 fragment, a homologue of the thyroglobulin type-1 domains, has been determined at 2.0 A resolution in complex with cathepsin L. The structure of the p41 fragment demonstrates a novel fold, consisting of two subdomains, each stabilized by disulfide bridges. The first subdomain is an alpha-helix-beta-strand arrangement, whereas the second subdomain has a predominantly beta-strand arrangement. The wedge shape and three-loop arrangement of the p41 fragment bound to the active site cleft of cathepsin L are reminiscent of the inhibitory edge of cystatins, thus demonstrating the first example of convergent evolution observed in cysteine protease inhibitors. However, the different fold of the p41 fragment results in additional contacts with the top of the R-domain of the enzymes, which defines the specificity-determining S2 and S1' substrate-binding sites. This enables inhibitors based on the thyroglobulin type-1 domain fold, in contrast to the rather non-selective cystatins, to exhibit specificity for their target enzymes. (+info)
C/EBPalpha regulates generation of C/EBPbeta isoforms through activation of specific proteolytic cleavage.
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C/EBPalpha and C/EBPbeta are intronless genes that can produce several N-terminally truncated isoforms through the process of alternative translation initiation at downstream AUG codons. C/EBPbeta has been reported to produce four isoforms: full-length 38-kDa C/EBPbeta, 35-kDa LAP (liver-enriched transcriptional activator protein), 21-kDa LIP (liver-enriched transcriptional inhibitory protein), and a 14-kDa isoform. In this report, we investigated the mechanisms by which C/EBPbeta isoforms are generated in the liver and in cultured cells. Using an in vitro translation system, we found that LIP can be generated by two mechanisms: alternative translation and a novel mechanism-specific proteolytic cleavage of full-length C/EBPbeta. Studies of mice in which the C/EBPalpha gene had been deleted (C/EBPalpha-/-) showed that the regulation of C/EBPbeta proteolysis is dependent on C/EBPalpha. The induction of C/EBPalpha in cultured cells leads to induced cleavage of C/EBPbeta to generate the LIP isoform. We characterized the cleavage activity in mouse liver extracts and found that the proteolytic cleavage activity is specific to prenatal and newborn livers, is sensitive to chymostatin, and is completely abolished in C/EBPalpha-/- animals. The lack of cleavage activity in the livers of C/EBPalpha-/- mice correlates with the decreased levels of LIP in the livers of these animals. Analysis of LIP production during liver regeneration showed that, in this system, the transient induction of LIP is dependent on the third AUG codon and most likely involves translational control. We propose that there are two mechanisms by which C/EBPbeta isoforms might be generated in the liver and in cultured cells: one that is determined by translation and a second that involves C/EBPalpha-dependent, specific proteolytic cleavage of full-length C/EBPbeta. The latter mechanism implicates C/EBPalpha in the regulation of posttranslational generation of the dominant negative C/EBPbeta isoform, LIP. (+info)
An antiviral mechanism of nitric oxide: inhibition of a viral protease.
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Although nitric oxide (NO) kills or inhibits the replication of a variety of intracellular pathogens, the antimicrobial mechanisms of NO are unknown. Here, we identify a viral protease as a target of NO. The life cycle of many viruses depends upon viral proteases that cleave viral polyproteins into individual polypeptides. NO inactivates the Coxsackievirus protease 3C, an enzyme necessary for the replication of Coxsackievirus. NO S-nitrosylates the cysteine residue in the active site of protease 3C, inhibiting protease activity and interrupting the viral life cycle. Substituting a serine residue for the active site cysteine renders protease 3C resistant to NO inhibition. Since cysteine proteases are critical for virulence or replication of many viruses, bacteria, and parasites, S-nitrosylation of pathogen cysteine proteases may be a general mechanism of antimicrobial host defenses. (+info)
Re-entering the translocon from the lumenal side of the endoplasmic reticulum. Studies on mutated carboxypeptidase yscY species.
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Misfolded or unassembled secretory proteins are retained in the endoplasmic reticulum (ER) and subsequently degraded by the cytosolic ubiquitin-proteasome system. This requires their retrograde transport from the ER lumen into the cytosol, which is mediated by the Sec61 translocon. It had remained a mystery whether ER-localised soluble proteins are at all capable of re-entering the Sec61 channel de novo or whether a permanent contact of the imported protein with the translocon is a prerequisite for retrograde transport. In this study we analysed two new variants of the mutated yeast carboxypeptidase yscY, CPY*: a carboxy-terminal fusion protein of CPY* and pig liver esterase and a CPY* species carrying an additional glycosylation site at its carboxy-terminus. With these constructs it can be demonstrated that the newly synthesised CPY* chain is not retained in the translocation channel but reaches its ER lumenal side completely. Our data indicate that the Sec61 channel provides the essential pore for protein transport through the ER membrane in either direction; persistent contact with the translocon after import seems not to be required for retrograde transport. (+info)
Oligosaccharide modification in the early secretory pathway directs the selection of a misfolded glycoprotein for degradation by the proteasome.
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The role of conformation-based quality control in the early secretory pathway is to eliminate misfolded polypeptides and unassembled multimeric protein complexes from the endoplasmic reticulum, ensuring the deployment of only functional molecules to distal sites. The intracellular fate of terminally misfolded human alpha1-antitrypsin was examined in hepatoma cells to identify the functional role of asparagine-linked oligosaccharide modification in the selection of glycoproteins for degradation by the cytosolic proteasome. Proteasomal degradation required physical interaction with the molecular chaperone calnexin. Altered sedimentation of intracellular complexes following treatment with the specific proteasome inhibitor lactacystin, and in combination with mannosidase inhibition, revealed that the removal of mannose from attached oligosaccharides abrogates the release of misfolded alpha1-antitrypsin from calnexin prior to proteasomal degradation. Intracellular turnover was arrested with kifunensine, implicating the participation of endoplasmic reticulum mannosidase I in the disposal process. Accelerated degradation occurred in a mannosidase-independent manner and was arrested by lactacystin, in response to the posttranslational inhibition of glucosidase II, demonstrating that the attenuated removal of glucose from attached oligosaccharides functions as the underlying rate-limiting step in the proteasome-mediated pathway. A model is proposed in which the removal of mannose from multiple attached oligosaccharides directs calnexin in the selection of misfolded alpha1-antitrypsin for degradation by the proteasome. (+info)
Possible involvement of proteasomes (prosomes) in AUUUA-mediated mRNA decay.
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We have identified a cellular target for proteasomal endonuclease activity. Thus, 20 S proteasomes interact with the 3'-untranslated region of certain cytoplasmic mRNAs in vivo, and 20 S proteasomes isolated from Friend leukemia virus-infected mouse spleen cells were found to be associated with a mRNA fragment showing great homology to the 3'-untranslated region of tumor necrosis factor-beta mRNA that contains AUUUA sequences. We furthermore demonstrate that 20 S proteasomes destabilize oligoribonucleotides corresponding to the 3'-untranslated region of tumor necrosis factor-alpha, creating a specific cleavage pattern. The cleavage reaction is accelerated with increasing number of AUUUA motifs, and major cleavage sites are localized at the 5' side of the A residues. These results strongly suggest that 20 S proteasomes could be involved in the destabilization of cytokine mRNAs such as tumor necrosis factor mRNAs and other short-lived mRNAs containing AUUUA sequences. (+info)
Mechanisms for generating the autonomous cAMP-dependent protein kinase required for long-term facilitation in Aplysia.
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The formation of a persistently active cAMP-dependent protein kinase (PKA) is critical for establishing long-term synaptic facilitation (LTF) in Aplysia. The injection of bovine catalytic (C) subunits into sensory neurons is sufficient to produce protein synthesis-dependent LTF. Early in the LTF induced by serotonin (5-HT), an autonomous PKA is generated through the ubiquitin-proteasome-mediated proteolysis of regulatory (R) subunits. The degradation of R occurs during an early time window and appears to be a key function of proteasomes in LTF. Lactacystin, a specific proteasome inhibitor, blocks the facilitation induced by 5-HT, and this block is rescued by injecting C subunits. R is degraded through an allosteric mechanism requiring an elevation of cAMP coincident with the induction of a ubiquitin carboxy-terminal hydrolase. (+info)
Constitutive degradation of PML/RARalpha through the proteasome pathway mediates retinoic acid resistance.
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PML/RARalpha is the leukemogenetic protein of acute promyelocytic leukemia (APL). Treatment with retinoic acid (RA) induces degradation of PML/RARalpha, differentiation of leukaemic blasts, and disease remission. However, RA resistance arises during RA treatment of APL patients. To investigate the phenomenon of RA resistance in APL, we generated RA-resistant sublines from APL-derived NB4 cells. The NB4.007/6 RA-resistant subline does not express the PML/RARalpha protein, although its mRNA is detectable at levels comparable to those of the parental cell line. In vitro degradation assays showed that the half-life of PML/RARalpha is less than 30 minutes in NB4.007/6 and longer than 3 hours in NB4. Treatment of NB4.007/6 cells with the proteasome inhibitors LLnL and lactacystin partially restored PML/RARalpha protein expression and resulted in a partial release of the RA-resistant phenotype. Similarly, forced expression of PML/RARalpha, but not RARalpha, into the NB4/007.6 cells restored sensitivity to RA treatment to levels comparable to those of the NB4 cells. These results indicate that constitutive degradation of PML/RARalpha protein may lead to RA resistance and that PML/RARalpha expression is crucial to convey RA sensitivity to APL cells. (+info)