Improving the turnaround time of molecular profiling for advanced non-small cell lung cancer: Outcome of a new algorithm integrating multiple approaches.

BACKGROUND: Molecular tumor profiling to identify oncogenic drivers and actionable mutations has a profound impact on how lung cancer is treated. Especially in the subgroup of non-small cell lung cancer (NSCLC), molecular testing for certain mutations is crucial in daily clinical practice and is recommended by international guidelines. To date, a standardized approach to identify druggable genetic alterations are lacking. We have developed and implemented a new diagnostic algorithm to harmonize the molecular testing of NSCLC. PATIENTS AND METHODS: In this retrospective analysis, we reviewed 119 patients diagnosed with NSCLC at the University Hospital Zurich. Tumor samples were analyzed using our standardized diagnostic algorithm: After the histological diagnosis was made, tissue samples were further analyzed by immunohistochemical stainings as well as the real-time PCR test Idylla™. Extracted DNA was further utilized for comprehensive genomic profiling (FoundationOne®CDx, F1CDx). RESULTS: Out of the 119 patients were included in this study, 100 patients were diagnosed with non-squamous NSCLC (nsqNSCLC) and 19 with squamous NSCLC (sqNSCLC). The samples from the nsqNSCLC patients underwent testing by Idylla™ and were evaluated by immunohistochemistry (IHC). F1CDx analysis was run on 67 samples and 46 potentially actionable genomic alterations were detected. Ten patients received the indicated targeted treatment. The median time to test results was 4 days for the Idylla test, 5 days for IHC and 13 days for the F1CDx. CONCLUSION: In patients with NSCLC, the implementation of a standardized molecular testing algorithm provided information on predictive markers for NSCLC within a few working days. The implementation of broader genomic profiling led to the identification of actionable targets, which would otherwise not have been discovered.

The Role of the Liquid Biopsy in Decision-Making for Patients with Non-Small Cell Lung Cancer.

Liquid biopsy is a rapidly emerging tool of precision oncology enabling minimally invasive molecular diagnostics and longitudinal monitoring of treatment response. For the clinical management of advanced stage lung cancer patients, detection and quantification of circulating tumor DNA (ctDNA) is now widely adopted into clinical practice. Still, interpretation of results and validation of ctDNA-based treatment decisions remain challenging. We report here our experience implementing liquid biopsies into the clinical management of lung cancer. We discuss advantages and limitations of distinct ctDNA assay techniques and highlight our approach to the analysis of recurrent molecular alterations found in lung cancer. Moreover, we report three exemplary clinical cases illustrating the complexity of interpreting liquid biopsy results in clinical practice. These cases underscore the potential and current limitations of liquid biopsy, focusing on the difficulty of interpreting discordant findings. In our view, despite all current limitations, the analysis of ctDNA in lung cancer patients is an essential and highly versatile complementary diagnostic tool for the clinical management of lung cancer patients in the era of precision oncology.

Prognostic relevance of lactate dehydrogenase and serum S100 levels in stage IV melanoma with known BRAF mutation status.

BACKGROUND: Activating mutations of BRAF provide an important treatment target in patients with melanoma. The prognostic role of several biochemical markers in relation to mutation status is not clear. OBJECTIVES: To analyse the prognostic significance of BRAF mutation in patients with melanoma and correlate it to different markers. METHODS: In total, 162 patients with stage IV melanoma and known BRAF mutation status were included. Clinical, histopathological and laboratory information was collected and compared between patients with BRAF mutant (BRAFm) and wild-type (BRAFwt) melanoma at the time of first distant metastasis. RESULTS: In total, 88 patients (54%) had BRAFm melanoma (V600E/V600K). At the first distant metastasis, S100B levels in BRAFm patients were more frequently elevated (P = 0·01) and significantly higher (P = 0·02). Median overall survival (mOS) was significantly longer in BRAFwt patients with normal compared with patients with elevated S100B levels (P < 0·01). In BRAFm melanoma, elevated S100B levels showed no prognostic influence (P = 0·18). Elevated lactate dehydrogenase (LDH) levels had a significantly negative impact on mOS in both groups. mOS was increased for BRAFm patients treated with a BRAF inhibitor (BRAFi) compared with BRAFm patients not receiving BRAFi (P = 0·01). No difference in mOS between BRAFm patients who did not receive BRAFi treatment and BRAFwt patients was observed. CONCLUSIONS: Better mOS was observed in BRAFm patients treated with BRAFi. BRAFm patients not treated with BRAFi show similar survival curves to BRAFwt patients. Elevated LDH is a BRAF-independent prognostic parameter; S100B has prognostic significance in BRAFwt melanoma only.

TLR9 triggering in Burkitt's lymphoma cell lines suppresses the EBV BZLF1 transcription via histone modification.

Endemic Burkitt's lymphoma (BL) is considered to preferentially develop in equatorial Africa because of chronic co-infection with Epstein-Barr virus (EBV) and the malaria pathogen Plasmodium falciparum. The interaction and contribution of both pathogens in the oncogenic process are poorly understood. Earlier, we showed that immune activation with a synthetic Toll-like receptor 9 (TLR9) ligand suppresses the initiation of EBV lytic replication in primary human B cells. In this study we investigate the mechanism involved in the suppression of EBV lytic gene expression in BL cell lines. We show that this suppression is dependent on functional TLR9 and MyD88 signaling but independent of downstream signaling elements, including phosphatidylinositol-3 kinase, mitogen-activated protein kinases and nuclear factor-kappaB. We identified TLR9 triggering resulting in histone modifications to negatively affect the activation of the promoter of EBV's master regulatory lytic gene BZLF1. Finally, we show that P. falciparum hemozoin, a natural TLR9 ligand, suppresses induction of EBV lytic gene expression in a dose-dependent manner. Thus, we provide evidence for a possible interaction between P. falciparum and EBV at the B-cell level and the mechanism involved in suppressing lytic and thereby reinforcing latent EBV that has unique oncogenic potential.

Lysine 188 substitutions convert the pattern of proteasome activation by REGgamma to that of REGs alpha and beta.

11S REGs (PA28s) are multimeric rings that bind proteasomes and stimulate peptide hydrolysis. Whereas REGalpha activates proteasomal hydrolysis of peptides with hydrophobic, acidic or basic residues in the P1 position, REGgamma only activates cleavage after basic residues. We have isolated REGgamma mutants capable of activating the hydrolysis of fluorogenic peptides diagnostic for all three active proteasome beta subunits. The most robust REGgamma specificity mutants involve substitution of Glu or Asp for Lys188. REGgamma(K188E/D) variants are virtually identical to REGalpha in proteasome activation but assemble into less stable heptamers/hexamers. Based on the REGalpha crystal structure, Lys188 of REGgamma faces the aqueous channel through the heptamer, raising the possibility that REG channels function as substrate-selective gates. However, covalent modification of proteasome chymotrypsin-like subunits by 125I-YL3-VS demonstrates that REGgamma(K188E)'s activation of all three proteasome active sites is not due to relaxed gating. We propose that decreased stability of REGgamma(K188E) heptamers allows them to change conformation upon proteasome binding, thus relieving inhibition of the CT and PGPH sites normally imposed by the wild-type REGgamma molecule.

Molecular dissection of the 11S REG (PA28) proteasome activators.

The proteasome activators known as 11S REG or PA28 were discovered about 10 years ago. They are homo- or heteroheptameric rings that bind to the ends of 20S proteasomes and activate cleavage of peptides but not folded proteins. In this article, we focus on structural features of three homologous REG subunits (termed alpha, beta, gamma) that contribute to their oligomerization, proteasome binding and proteasome activation. We review a number of published studies on the biochemical properties of REGs and present new results in which N-terminal sequences and sequences flanking REG activation loops have been exchanged between homologs. Characterization of these chimeras and previously constructed C-terminal chimeras reveal that N-terminal and loop flanking sequences affect oligomerization, whereas C-terminal sequences are essential for proteasome binding. None of these regions is responsible for the broad activation specificity of REGs alpha/beta versus the narrow specificity of REGgamma. Rather, mutation in a single residue lining the channel through the REGgamma heptamer changes the activation property of the gamma homolog to match that of REGs alpha and beta.

Substrate specificity of the human proteasome.

BACKGROUND: Regulated proteolysis by the proteasome is crucial for a broad array of cellular processes, from control of the cell cycle to production of antigens. RESULTS: The rules governing the N-terminal primary and extended substrate specificity of the human 20S proteasome in the presence or absence of 11S proteasome activators (REGalpha/beta and REGgamma) have been elaborated using activity-based proteomic library tools. CONCLUSIONS: The 11S proteasome activators are shown to be important for both increasing the activity of the 20S proteasome and for altering its cleavage pattern and substrate specificity. These data also establish that the extended substrate specificity is an important factor for proteasomal cleavage. The specificities observed have features in common with major histocompatibility complex (MHC) class I ligands and can be used to improve the prediction of MHC class I restricted cytotoxic T-cell responses.

The proteasome activator 11 S REG or PA28: chimeras implicate carboxyl-terminal sequences in oligomerization and proteasome binding but not in the activation of specific proteasome catalytic subunits.

The REG homologs, alpha, beta and gamma, activate mammalian proteasomes in distinct ways. REGalpha and REGbeta activate the trypsin-like, chymotrypsin-like and peptidylglutamyl-preferring active sites, whereas REGgamma only activates the proteasome's trypsin-like subunit. The three REG homologs differ in carboxyl-terminal sequences that are located next to activation loops on their proteasome binding surface. To assess the importance of these carboxyl-terminal sequences in the activation of specific proteasome beta catalytic subunits, we characterized chimeras in which 8 or 12 residues were exchanged among the three proteins. Like the wild-type molecule, REGalpha chimeras activated all three proteasome catalytic subunits regardless of the carboxyl-terminal sequence. However, REGalpha-beta chimeras activated the proteasome at lower concentrations than wild-type REGalpha and higher levels of REGalpha-gamma chimeras were needed for maximal activation because exchanged carboxyl-terminal sequences can stabilize (REGalpha-beta) or destabilize (REGalpha-gamma) the REGalpha heptamer. REGgamma chimeras were equivalent to REGgamma in their activation properties, but they bound the proteasome less tightly than the wild-type molecule. REGbeta chimeras also bound the proteasome more weakly than wild-type REGbeta and were virtually unable to activate it. Our findings demonstrate that the carboxyl-terminal sequences of REG subunits can affect heptamer stability and proteasome affinity, but they do not determine which proteasome beta subunits become activated.

Subcellular localization of proteasomes and their regulatory complexes in mammalian cells.

Proteasomes can exist in several different molecular forms in mammalian cells. The core 20S proteasome, containing the proteolytic sites, binds regulatory complexes at the ends of its cylindrical structure. Together with two 19S ATPase regulatory complexes it forms the 26S proteasome, which is involved in ubiquitin-dependent proteolysis. The 20S proteasome can also bind 11S regulatory complexes (REG, PA28) which play a role in antigen processing, as do the three variable gamma-interferon-inducible catalytic beta-subunits (e.g. LMP7). In the present study, we have investigated the subcellular distribution of the different forms of proteasomes using subunit specific antibodies. Both 20S proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic and microsomal preparations isolated from rat liver. LMP7 was enriched approximately two-fold compared with core alpha-type proteasome subunits in the microsomal preparations. 20S proteasomes were more abundant than 26S proteasomes, both in liver and cultured cell lines. Interestingly, some significant differences were observed in the distribution of different subunits of the 19S regulatory complexes. S12, and to a lesser extent p45, were found to be relatively enriched in nuclear fractions from rat liver, and immunofluorescent labelling of cultured cells with anti-p45 antibodies showed stronger labelling in the nucleus than in the cytoplasm. The REG was found to be localized predominantly in the cytoplasm. Three- to six-fold increases in the level of REG were observed following gamma-interferon treatment of cultured cells but gamma-interferon had no obvious effect on its subcellular distribution. These results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells and, therefore, that the distribution is more complex than has been reported for yeast proteasomes.

Recognition of the polyubiquitin proteolytic signal.

Polyubiquitin chains linked through Lys48 are the principal signal for targeting substrates to the 26S proteasome. Through studies of structurally defined, polyubiquitylated model substrates, we show that tetraubiquitin is the minimum signal for efficient proteasomal targeting. The mechanism of targeting involves a simple increase in substrate affinity that is brought about by autonomous binding of the polyubiquitin chain. Assigning the proteasomal signaling function to a specific polymeric unit explains how a single ubiquitin can act as a functionally distinct signal, for example in endocytosis. The properties of the substrates studied here implicate substrate unfolding as a kinetically dominant step in the proteolysis of properly folded proteins, and suggest that extraproteasomal chaperones are required for efficient degradation of certain proteasome substrates.

Mapping subunit contacts in the regulatory complex of the 26 S proteasome. S2 and S5b form a tetramer with ATPase subunits S4 and S7.

The 19 S regulatory complex (RC) of the 26 S proteasome is composed of at least 18 different subunits, including six ATPases that form specific pairs S4-S7, S6-S8, and S6'-S10b in vitro. One of the largest regulatory complex subunits, S2, was translated in reticulocyte lysate containing [(35)S]methionine and used to probe membranes containing SDS-polyacrylamide gel electrophoresis separated RC subunits. S2 bound to two ATPases, S4 and S7. Association of S2 with regulatory complex subunits was also assayed by co-translation and sedimentation. S2 formed an immunoprecipitable heterotrimer upon co-translation with S4 and S7. The non-ATPase S5b also formed a ternary complex with S4 and S7 and the three proteins assembled into a tetramer with S2. Neither S2 nor S5b formed complexes with S6'-S10b dimers or with S6-S8 oligomers. The use of chimeric ATPases demonstrated that S2 binds the NH(2)-terminal region of S4 and the COOH-terminal two-thirds of S7. Conversely, S5b binds the COOH-terminal two-thirds of S4 and to S7's NH(2)-terminal region. The demonstrated association of S2 with ATPases in the mammalian 19 S regulatory complex is consistent with and extends the recent finding that the yeast RC is composed of two subcomplexes, the lid and the base (Glickman, M. H., Rubin, D. M., Coux, O., Wefes, I., Pfeifer, G., Cejka, Z., Baumeister, W., Fried, V. A., and Finley, D. (1998) Cell 94, 615-623).

The proteasome activator 11 S REG (PA28) and class I antigen presentation.

There are two immune responses in vertebrates: humoral immunity is mediated by circulating antibodies, whereas cytotoxic T lymphocytes (CTL) confer cellular immunity. CTL lyse infected cells upon recognition of cell-surface MHC Class I molecules complexed with foreign peptides. The displayed peptides are produced in the cytosol by degradation of host proteins or proteins from intracellular pathogens that might be present. Proteasomes are cylindrical multisubunit proteases that generate many of the peptides eventually transferred to the cell surface for immune surveillance. In mammalian proteasomes, six active sites face a central chamber. As this chamber is sealed off from the enzyme's surface, there must be mechanisms to promote entry of substrates. Two protein complexes have been found to bind the ends of the proteasome and activate it. One of the activators is the 19 S regulatory complex of the 26 S proteasome; the other activator is '11 S REG' [Dubiel, Pratt, Ferrell and Rechsteiner (1992) J. Biol. Chem. 267, 22369-22377] or 'PA28' [Ma, Slaughter and DeMartino (1992) J. Biol. Chem. 267, 10515-10523]. During the past 7 years, our understanding of the structure of REG molecules has increased significantly, but much less is known about their biological functions. There are three REG subunits, namely alpha, beta and gamma. Recombinant REGalpha forms a ring-shaped heptamer of known crystal structure. 11 S REG is a heteroheptamer of alpha and beta subunits. REGgamma is also presumably a heptameric ring, and it is found in the nuclei of the nematode work Caenorhabditis elegans and higher organisms, where it may couple proteasomes to other nuclear components. REGalpha and REGbeta, which are abundant in vertebrate immune tissues, are located mostly in the cytoplasm. Synthesis of REG alpha and beta subunits is induced by interferon-gamma, and this has led to the prevalent hypothesis that REG alpha/beta hetero-oligomers play an important role in Class I antigen presentation. In the present review we focus on the structural properties of REG molecules and on the evidence that REGalpha/beta functions in the Class I immune response.

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.

Discrimination between ubiquitin-dependent and ubiquitin-independent proteolytic pathways by the 26S proteasome subunit 5a.

The 26S proteasome subunit 5a binds polyubiquitin chains and has previously been shown to inhibit the degradation of mitotic cyclins. Presumably inhibition results from S5a binding and preventing recognition of Ub-cyclin conjugates by the 26S proteasome. Here we show that S5a does not inhibit the degradation of full-length ornithine decarboxylase (ODC) consistent with previous reports that the enzyme is degraded in an antizyme-dependent, but ubiquitin-independent reaction. S5a does, however, inhibit degradation of short ODC translation products generated by internal initiation events. Because in vitro translation often produces some shortened products, the existence of ubiquitin conjugated to a 35S-labeled protein is not necessarily evidence that the full-length protein is a substrate of the Ub-dependent proteolytic pathway.

Assembly of the regulatory complex of the 26S proteasome.

The 19S regulatory complex (RC) of 26S proteasomes is a 900-1000 kDa particle composed of 18 distinct subunits (S1-S15) ranging in molecular mass from 25 to 110 kDa. This particle confers ATP-dependence and polyubiquitin (polyUb) recognition to the 26S proteasome. The symmetry and homogenous structure of the proteasome contrasts sharply with the remarkable complexity of the RC. Despite the fact that the primary sequences of all the subunits are now known, insight has been gained into the function of only eight subunits. The six ATPases within the RC constitute a subfamily (S4-like ATPases) within the AAA superfamily and we have shown that they form specific pairs in vitro. We have now determined that putative coiled-coils within the variable N-terminal regions of these proteins are likely to function as recognition elements that direct the proper placement of the ATPases within the RC. We have also begun mapping putative interactions between non-ATPase subunits and S4-like ATPases. These studies have allowed us to build a model for the specific arrangement of 9 subunits within the human regulatory complex. This model agrees with recent findings by Glickman et al. who have reported that two subcomplexes, termed the base and the lid, form the RC of budding yeast 26S proteasomes.

Identification, molecular cloning, and characterization of subunit 11 of the human 26S proteasome.

We sequenced five peptides from subunit 11 (S11), a 43 kDa protein of the human 26S proteasome, and used this information to clone its cDNA. The S11 cDNA encodes a 376 amino acid protein with a pI of 5.6 and a molecular mass of 42.9 kDa. Translation of S11 RNA in the presence of [35S]methionine produces a radiolabeled protein that co-migrates with S11 of the human 26S proteasome on SDS-PAGE. Polyclonal antiserum made against recombinant S11 recognizes a protein of the same size in extracts of bacteria expressing S11 and in purified 26S proteasomes from human red blood cells or rabbit reticulocytes. The S11 sequence does not contain motifs that suggest a biological function. S11 is, however, the human homolog of Rpn9, a recently identified subunit of the yeast 26S proteasome.

Proteasome activator 11S REG or PA28: recombinant REG alpha/REG beta hetero-oligomers are heptamers.

The proteasome activator 11S REG or PA28 is a conical molecule composed of two homologous subunits, REG alpha and REG beta. Recombinant REG alpha forms a heptamer, whereas recombinant REG beta is a monomer. When mixed with REG beta, a monomeric REG alpha mutant (N50Y) forms an active hetero-oligomer in which the molar ratio of REG beta to REG alpha(N50Y) is close to 1.3. This apparent stoichiometry is consistent with the REG alpha(N50Y)/REG beta hetero-oligomer being a heptamer composed of three alpha and four beta subunits. Chemical cross-linking of the alpha/beta oligomers revealed the presence of REG alpha-REG beta and REG beta-REG beta dimers, but REG alpha-REG alpha dimers were not detected. The mass of the REG alpha(N50Y)/REG beta hetero-oligomer determined by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) is 194 871 +/- 40 Da in good agreement with the theoretical mass of 194 856 Da for an alpha 3 beta 4 heptamer. Hexamers were not observed in the mass spectrum. For wild-type REG subunits coexpressed in bacteria cells at an apparent beta/alpha molar ratio of approximately 1.2, the resulting hetero-oligomers observed by ESI-TOF MS were again predominantly alpha 3 beta 4 heptamers, with trace amounts of alpha 4 beta heptamers also present. On the other hand, the mass spectrum contained a mixture of alpha 7, alpha 6 beta 1, alpha 5 beta 2, and alpha 4 beta 3 heptamers when the REG beta/REG alpha ratio was 0.1. Thus, formation of heptamers is an intrinsic property of recombinant REG alpha and REG beta subunits. On the basis of these results, we propose that 11S REG purified directly from eukaryotic cells is also heptameric, likely alpha 3 beta 4 or a mixture of alpha 3 beta 4 and alpha 4 beta 3 species.

The proteasome activator 11 S regulator or PA28. Contribution By both alpha and beta subunits to proteasome activation.

The proteasome 11 S regulator (REG) consists of two homologous subunits, REGalpha and REGbeta. Each subunit is capable of activating the proteasome, and when combined, they form superactive REGalpha/REGbeta complexes. We have previously shown that a highly conserved loop in the REGalpha crystal structure is critical for proteasome activation. We now show that hetero-oligomers formed from REGalpha activation loop mutants and wild-type REGbeta or vice versa are partially active. By contrast, hetero-oligomers bearing mutations in the activation loops of REGalpha and REGbeta subunits are inactive, demonstrating that both alpha and beta subunits contribute to proteasome activation. We have also characterized REG proteins with mutations near or at their C termini. Partially active REGalpha(Y249C) and REGalpha(M247V) and an inactive REGalpha subunit bearing five additional C-terminal amino acids formed active hetero-oligomers with REGbeta. REGbeta subunits lacking 1, 2, or 9 C-terminal amino acids did not bind or activate the proteasome, but each of these mutants formed partially active hetero-oligomers with the monomer REGalpha(N50Y). However, hetero-oligomers formed from REG subunits lacking the last 14 amino acids were unable to bind the proteasome. Thus, C-terminal regions of both alpha and beta subunits are required for hetero-oligomers to bind the proteasome.

Proteasome activation by REG molecules lacking homolog-specific inserts.

The peptidase activities of eukaryotic proteasomes are markedly activated by the 11 S REG or PA28. The three identified REG subunits, designated alpha, beta, and gamma, differ significantly in sequence over a short span of 15-30 amino acids that we call homolog-specific inserts. These inserts were deleted from each REG to produce the mutant proteins REGalphaDeltai, REGbetaDeltai, and REGgammaDeltai. The purified recombinant proteins were then tested for their ability to oligomerize and activate the proteasome. Both REGalphaDeltai and REGgammaDeltai formed apparent heptamers and activated human red cell proteasomes to the same extent as their full-length counterparts. By contrast, REGbetaDeltai exhibited, at low protein concentrations, reduced proteasome activation when compared with the wild-type REGbeta protein. REGbetaDeltai was able to form hetero-oligomers with a single site, monomeric REGalpha mutant and with REGalphaDeltai. At low concentrations, the REGalphaDeltai/REGbetaDeltai hetero-oligomers stimulated the proteasome less than REGalpha/REGbeta oligomers formed from wild-type subunits, and the reduced activation by REGalphaDeltai/REGbetaDeltai was due to removal of the REGbeta insert, not the REGalpha insert. These studies demonstrate that the REGalpha and REGgamma inserts play virtually no role in oligomerization or in proteasome activation. By contrast, removal of REGbeta insert reduces binding of this subunit and REGalpha/REGbeta oligomers to proteasomes. On the whole, however, our findings show that REG inserts are not required for binding and activating the proteasome. We speculate that they serve to localize REG-proteasome complexes within cells, possibly by binding components in endoplasmic reticulum membranes.