A. Jennifer Rivett

Rapid caspase‐3 activation during apoptosis revealed using fluorescence‐resonance energy transfer

Caspase‐3 is a crucial component of the apoptotic machinery in many cell types. Here, we report the timescale of caspase‐3 activation in single living cells undergoing apoptosis. This was achieved by measuring the extent of fluorescence resonance energy transfer within a recombinant substrate containing cyan fluorescent protein (CFP) linked by a short peptide possessing the caspase‐3 cleavage sequence, DEVD, to yellow fluorescent protein (YFP; i.e. CFP–DEVD–YFP). We demonstrate that, once initiated, the activation of caspase‐3 is a very rapid process, taking 5 min or less to reach completion. Furthermore, this process occurs almost simultaneously with a depolarization of the mitochondrial membrane potential. These events occur just prior to the characteristic morphological changes associated with apoptosis. Our results clearly demonstrate that, once initiated, the commitment of cells to apoptosis is a remarkably rapid event when visualized at the single cell level.

INTRACELLULAR DISTRIBUTION OF PROTEASOMES

Proteasomes are large multicatalytic proteinase complexes which are responsible for the selective degradation of cellular proteins and the production of peptides for antigen presentation. Proteasomes are localized both in the nucleus and in the cytoplasm, where some are associated with the endoplasmic reticulum membrane. Recent studies have shown differences in the localization of proteasome subpopulations, demonstrated the functional importance of endoplasmic reticulum-associated proteasomes and investigated the role of putative nuclear localization signals and tyrosine phosphorylation on proteasome transport into the nucleus.

Changes in the proteolytic activities of proteasomes and lysosomes in human fibroblasts produced by serum withdrawal, amino-acid deprivation and confluent conditions.

The contribution of the main proteolytic pathways to the degradation of long-lived proteins in human fibroblasts grown under different conditions was investigated. The effects of various commonly used pharmacological inhibitors of protein degradation were first analysed in detail. By choosing specific inhibitors of lysosomes and proteasomes, it was observed that together both pathways accounted for 80% or more of the degradation of cell proteins. With lysosomal inhibitors, it was found that serum withdrawal or amino-acid deprivation strongly stimulated macroautophagy but not other lysosomal pathways, whereas confluent conditions had no effect on macroautophagy and slightly activated other lysosomal pathways. Prolonged (24 h) serum starvation of confluent cultures strongly decreased the macroautophagic pathway, whereas the activity of other lysosomal pathways increased. These changes correlated with electron microscopic observations and morphometric measurements of lysosomes. With proteasomal inhibitors, it was found that, in exponentially growing cells in the absence of serum, activity of the ubiquitin-proteasome pathway increases, whereas under confluent conditions the contribution (in percentage) of proteasomes to degradation decreases, especially in cells deprived of amino acids. Interestingly, in confluent cells, the levels of two components of the 19 S regulatory complex and those of an interchangeable beta-subunit decreased. This was associated with a marked increase in the levels of components of PA28-immunoproteasomes. Thus confluent conditions affect proteasomes in a way that resembles treatment with interferon-gamma. Altogether, these results show that the activity of the various proteolytic pathways depends on the growth conditions of cells and will be useful for investigation of the specific signals that control their activity.

Phosphorylation of 20S proteasome alpha subunit C8 (alpha7) stabilizes the 26S proteasome and plays a role in the regulation of proteasome complexes by gamma-interferon.

In animal cells there are several regulatory complexes which interact with 20S proteasomes and give rise to functionally distinct proteasome complexes. gamma-Interferon upregulates three immuno beta catalytic subunits of the 20S proteasome and the PA28 regulator, and decreases the level of 26S proteasomes. It also decreases the level of phosphorylation of two proteasome alpha subunits, C8 (alpha7) and C9 (alpha3). In the present study we have investigated the role of phosphorylation of C8 by protein kinase CK2 in the formation and stability of 26S proteasomes. An epitope-tagged C8 subunit expressed in mammalian cells was efficiently incorporated into both 20S proteasomes and 26S proteasomes. Investigation of mutants of C8 at the two known CK2 phosphorylation sites demonstrated that these are the two phosphorylation sites of C8 in animal cells. Although phosphorylation of C8 was not absolutely essential for the formation of 26S proteasomes, it did have a substantial effect on their stability. Also, when cells were treated with gamma-interferon, there was a marked decrease in phosphorylation of C8, a decrease in the level of 26S proteasomes, and an increase in immunoproteasomes and PA28 complexes. These results suggest that the down-regulation of 26S proteasomes after gamma-interferon treatment results from the destabilization that occurs after dephosphorylation of the C8 subunit.

Regulation of proteasome complexes by γ-interferon and phosphorylation

Abstract Proteasomes play a major role in non-lysosomal proteolysis and also in the processing of proteins for presentation by the MHC class I pathway. In animal cells they exist in several distinct molecular forms which contribute to the different functions. 26S proteasomes contain the core 20S proteasome together with two 19S regulatory complexes. Alternatively, PA28 complexes can bind to the ends of the 20S proteasome to form PA28-proteasome complexes and PA28-proteasome-19S hybrid complexes have also been described. Immunoproteasome subunits occur in 26S proteasomes as well as in PA28-proteasome complexes. We have found differences in the subcellular distribution of the different forms of proteasomes. The γ-interferon inducible PA28 α and β subunits are predominantly located in the cytoplasm, while 19S regulatory complexes (present at significant levels only in 26S complexes) are present in the nucleus as well as in the cytoplasm. Immunoproteasomes are greatly enriched at the endoplasmic reticulum (ER) where they may facilitate the generation of peptides for transport into the lumen of the ER. We have also investigated the effects of γ-interferon on the levels and subcellular distribution of inducible subunits and regulator subunits. In each case γ-interferon was found to increase the level but not to alter the distribution. Several subunits of proteasomes are phosphorylated including alpha subunits C8 (α7) and C9 (α3), and ATPase subunit S4 (rpt2). Our studies have shown that γ-interferon treatment decreases the level of phosphorylation of proteasomes. We have investigated the role of phosphorylation of C8 by casein kinase II by site directed mutagenesis. The results demonstrate that phosphorylation at either one of the two sites is essential for the association of 19S regulatory complexes and that the ability to undergo phosphorylation at both sites gives the most efficient incorporation of C8 into the 26S proteasome.

LMP2+ proteasomes are required for the presentation of specific antigens to cytotoxic T lymphocytes

BACKGROUND Major histocompatibility complex (MHC) class I molecules present short peptides generated by intracellular protein degradation to cytotoxic T lymphocytes (CTL). The multisubunit, non-lysosomal proteinases known as proteasomes have been implicated in the generation of these peptides. Two interferon-gamma (IFN-gamma)-inducible proteasome subunits, LMP2 and LMP7, are encoded within the MHC gene cluster in a region associated with antigen presentation. The incorporation of these LMP subunits into proteasomes may alter their activity so as to favour the generation of peptides able to bind to MHC class I molecules. It has been difficult, however, to demonstrate a specific requirement for LMP2 or LMP7 in the presentation of peptide epitopes to CTL. RESULTS We describe a T-cell lymphoma, termed SP3, that displays a novel selective defect in MHC class I-restricted presentation of influenza virus antigens. Of the MHC-encoded genes implicated in the class I pathway, only LMP2 is underexpressed in SP3 cells. Expression of IFN-gamma in transfected SP3 cells simultaneously restores LMP2 expression and antigen presentation to CTL. Expression of antisense-LMP2 mRNA in these IFN-gamma-transfected cells selectively represses antigen recognition and the induction of surface class I MHC expression. Moreover, the expression of this antisense-LMP2 mRNA in L929 fibroblast cells, which constitutively express LMP2 and have no presentation defect, blocks the presentation of the same influenza virus antigens that SP3 cells are defective in presenting. CONCLUSIONS Our results show that the LMP2 proteasome subunit can directly influence both MHC class I-restricted antigen presentation and class I surface expression.

[24] Multicatalytic endopeptidase complex: Proteasome

Publisher Summary This chapter describes assay, purification, and properties of proteasome. The multicatalytic endopeptidase complex or proteasome is a 700-kDa multisubunit enzyme complex that is widely distributed in eukaryotic cells. The multicatalytic endopeptidase complex, either by itself or as the catalytic core of the 26S proteinase complex, is assumed to play an important role in ubiquitin-dependent and ubiquitin independent nonlysosomal pathways of protein turnover, including the degradation of regulatory proteins and the processing of antigens for presentation by the major histocompatibility complex (MHC) class I pathway. Different substrates have been used to assay the endopeptidase activities of the complex purified from many different sources. Its substrates include proteins, peptides, and synthetic peptides and also the enzyme is active over a range of neutral to weakly alkaline pH values in a variety of different buffers. The 7-amino-4-methylcoumarin leaving group provides the most sensitive assay and these synthetic peptides are often used at concentrations below their K m values, which are in the range of 0.1-1 mM.

Interaction of U-box E3 ligase SNEV with PSMB4, the β7 subunit of the 20 S proteasome

Recognition of specific substrates for degradation by the ubiquitin-proteasome pathway is ensured by a cascade of ubiquitin transferases E1, E2 and E3. The mechanism by which the target proteins are transported to the proteasome is not clear, but two yeast E3s and one mammalian E3 ligase seem to be involved in the delivery of targets to the proteasome, by escorting them and by binding to the 19 S regulatory particle of the proteasome. In the present study, we show that SNEV (senescence evasion factor), a protein with in vitro E3 ligase activity, which is also involved in DNA repair and splicing, associates with the proteasome by directly binding to the beta7 subunit of the 20 S proteasome. Upon inhibition of proteasome activity, SNEV does not accumulate within the cells although its co-localization with the proteasome increases significantly. Since immunofluorescence microscopy also shows increased co-localization of SNEV with ubiquitin after proteasome inhibition, without SNEV being ubiquitinated by itself, we suggest that SNEV shows E3 ligase activity not only in vitro but also in vivo and escorts its substrate to the proteasome. Since the yeast homologue of SNEV, Prp19, also interacts with the yeast beta7 subunit of the proteasome, this mechanism seems to be conserved during evolution. Therefore these results support the hypothesis that E3 ligases might generally be involved in substrate transport to the proteasome. Additionally, our results provide the first evidence for a physical link between components of the ubiquitin-proteasome system and the spliceosome.

A pre‐existing protease is a common effector of thymocyte apoptosis mediated by diverse stimuli

Data from a number of model systems support a role for proteolysis in apoptotic cell death. Using immature rat thymocytes, we demonstrate that the inhibitors chloromethylketone (TLCK) and chloromethylketone (TPCK) have very different effects on apoptosis. TLCK inhibits apoptosis induced by diverse stimuli at an early stage prior to both DNA fragmentation and cytoplasmic changes. We show that the TLCK‐sensitive target is pre‐existing and not synthesized in response to apoptotic stimuli. The contrasting effects of TLCK and TPCK support the hypothesis that the TLCK target is a trypsin‐like protease which is a common effector of thymocyte apoptosis.

Proteasome response to interferon‐γ is altered in senescent human fibroblasts

We have investigated immunoproteasomes in human fibroblasts during replicative senescence. Unlike levels of constitutive proteasome catalytic subunits and 26S proteasome regulatory subunits, levels of immunosubunits did not decrease dramatically in senescent cells. However, the induction of immunosubunits by interferon‐γ (IFN‐γ) was lost in senescent cells. In contrast, levels of the 11S proteasome regulator, PA28, were increased by IFN‐γ even in senescent cells, and both immunosubunits and PA28 increased with the reversible growth arrest in confluent cell cultures. The results highlight differences in the mechanisms of regulation of immunoproteasomes compared to constitutive proteasomes and in the irreversible growth arrest of senescent cells compared to reversible contact‐induced growth arrest.