A pathway sensor for genome-wide screens of intracellular proteolytic cleavage
© Ketteler et al.; licensee BioMed Central Ltd. 2008
Received: 25 February 2008
Accepted: 3 April 2008
Published: 03 April 2008
Protein cleavage is a central event in many regulated biological processes. We describe a system for detecting intracellular proteolysis based on non-conventional secretion of Gaussia luciferase (GLUC). GLUC exits the cell without benefit of a secretory leader peptide, but can be anchored in the cell by fusion to β-actin. By including protease cleavage sites between GLUC and β-actin, proteolytic cleavage can be detected. Using this assay, we have identified regulators of autophagy, apoptosis and β-actin cleavage.
Advances in automation and the availability of genomic sequence information have led to the development of sophisticated cell-based assays for high-throughput screening of functional phenotypes . Most cell-based assays rely on fluorescent or luminescent reporters such as green fluorescent protein (GFP), secreted alkaline phosphatase (SEAP) or Photinus luciferase. Secreted luciferases offer many advantages over cellular reporter enzymes as they can be non-destructively harvested from cellular supernatants over time. Several secreted luciferases have been reported, from the marine copepods Gaussia princeps , and Metridia longa , the ostracod Vargula hilgendorfii , the decapod shrimp Oplophorus gracilirostris  and the ostracod crustacean Cypridina noctiluca . In addition, intracellular luciferases, such as from the sea pansy Renilla reniformis, can be engineered to be secreted and stable in the extra-cellular milieu .
A cDNA encoding G. princeps luciferase (GLUC) activity has recently been isolated and found to direct the synthesis of a 19.9 kDa protein that has utility as a bioluminescent reporter . GLUC can be used to monitor in vivo processes and can be easily harvested from biological fluids such as blood or urine . Assays based on GLUC activity have been used to study, among other topics, processing through the secretory pathway , the strength of signal peptides , endoplasmic reticulum (ER) stress , DNA hybridization , and protein-protein interaction using complementary fragments derived from the enzyme . By deletion of the signal peptide, a GLUC mutant has been engineered for monitoring in vivo gene expression; very low bioluminescence was detected in cell culture superanatants upon expression of this construct . However, overall bioluminescence of this construct was greatly reduced compared to wild-type GLUC . It has been noted that GLUC is secreted when fused to the ER retention signal KDEL, which has been attributed to changes in the protein conformation or processing in the ER and Golgi .
We have generated a GLUC variant that is secreted in the absence of a signal peptide. We present here a cell-based assay for the detection of general protease activity based on inducible luciferase secretion. GLUC can be anchored in cells by fusion to β-actin. Insertion of protease cleavage sites in a linker between β-actin and GLUC allows monitoring the cleavage of short peptides, as well as cleavage of native full-length proteins of any sequence inserted. We present GLUC-based reporter systems for monitoring apoptosis and autophagy and describe applications of this reporter in genome-wide screening approaches.
dNGLUC is secreted in the absence of a signal peptide
96.7 ± 27.6
30.5 ± 7.8
1.5 ± 0.4
Genes that induce release of dNGLUC activity in SN
Fold ratio SN/cells
14.2 ± 5.8
12.5 ± 2.3
12.1 ± 2.7
10.4 ± 2.5
5.5 ± 2.7
4.9 ± 1.8
4.8 ± 2.7
3.2 ± 1.1
In the course of the screen we also identified genes that induce release of dNGLUC from Actin-dNGLUC. Co-expression of the serine peptidase HTRA4 with ActindNGLUC or DEVDG2F yielded a 201.5-fold increase of GLUC activity in SN from cells expressing Actin-dNGLUC and a 110.8-fold increase from DEVDG2F, indicating that the caspase cleavage site is not required for liberation of luciferase activity (Figure 3b). Similarly, another family member, HTRA3, induced a 177.1-fold and 89.1-fold increase in GLUC activity in SN for Actin-dNGLUC and DEVDG2F, respectively. Caspases 8 and 9 induced a 9.5-fold and 15.0-fold increase of GLUC activity for DEVDG2F, but had no effect on Actin-dNGLUC. In accordance with previous reports that have identified HTRA2-mediated cleavage of β-actin by mass spectroscopy , these data support the view that HTRA3 and 4 cleave within the β-actin sequence. We therefore conclude that our assay also allows the detection of full-length protein cleavage under physiological conditions.
Non-conventional secretion of Gaussialuciferase
Protein secretion in most cells is mediated by signal sequences that target the nascent polypeptide chain of the elongating translation product to a secretory pore in the ER . Within the ER and the subsequent compartments of the Golgi apparatus, folding and post-translational modifications take place, and the mature, modified polypeptide is released into the extracellular space. A number of secreted proteins that do not utilize the ER membrane translocation machinery, such as fibroblast growth factor, coagulation factor XIII and interleukin-1β are secreted by a non-conventional secretory pathway . Different mechanisms for non-conventional secretion have been proposed , including lysosomal secretion for interleukin-1β , a plasma resident transporter for fibroblast growth factor 2  and cell injury for coagulation factor XIII . Two prevalent features of non-conventional secretion are the absence of a signal peptide and insensitivity to Brefeldin A . The precise mechanism of secretion is still poorly understood and the underlying molecular signals remain to be elucidated.
The luciferase release assay reported here relies on a non-conventional secretion of dNGLUC that is inhibited by Monensin and Brefeldin A. Monensin inhibits acidification of terminal compartments thought to lie immediately prior to extracellular release, whereas Brefeldin A inhibits ER-to-Golgi transport. The amino-terminal amino acid sequence of the deleted luciferase studied here does not fulfill the accepted criteria for a signal peptide . Because secretion is sensitive to treatment with Brefeldin A, we conclude that a previously unarticulated mechanism is responsible for the translocation of the polypeptide into the ER and/or Golgi. The molecular basis of this translocation, and subsequent passage through the terminal secretory apparatus, is presently under investigation. A Golgi-resident protein, GRASP, has been identified that is required for a non-conventional secretory pathway in Dictyostelium discoideum  and Drosophila melanogaster , and that is a candidate for mediating Brefeldin A-sensitive secretion of dNGLUC. Identification of GLUC mutants that are retained inside cells may help to identify the mechanism of non-conventional secretion.
A novel protease assay
The present assay system has several advantages over existing systems for measuring protease activity. Currently, protease cleavage sites can be inferred from comparison of primary sequences. The physiological relevance of predicted cleavage sites in a particular protein then can be assessed by further experimentation. Target motifs can be identified by analysis of protease action on peptide libraries, such as phage display libraries, positional-scanning libraries and mixture-based libraries . The identification of protein cleavage in the context of live cells can be achieved by mass spectroscopic analysis of cleavage products , but requires a complex experimental setup and is not amenable to high-throughput approaches. Other cell-based protease assays rely on generation of a fluorogenic substrate upon cleavage, but these assays are not genetically encoded, thus limiting their applicability in vivo. Some in vivo protease assays have been developed that exploit the properties of fluorescence resonance energy transfer (FRET) ; in these, the protease-mediated separation of a donor and acceptor fluorophore results in changes of the ratio of fluorescence intensities at different wavelengths . A major advantage of FRET-based methods is their ability to provide information about the sub-cellular localization of protease activity. However, FRET-based assays are frequently not highly sensitive, require a carefully characterized cohort of control samples in a single experiment and typically demand advanced instrumentation. To date there has been little use of FRET in genomic screening applications. In contrast, the assay system described here non-invasively measures protein cleavage over time in the context of the complex physiology of intact living cells, is compatible with high-throughput screening methodologies, and can be designed to monitor protease function with high specificity. The luciferase release system can detect cleavage of short peptides as well as cleavage of full-length proteins. Evaluation of actin-specific or non-specific screening hits can be identified and eliminated by secondary screening with a luciferase fusion bearing a mutated version of the protease cleavage motif to be investigated. It has previously been established that GLUC secretion is proportional to cell number . Differences in cell number as well as variation in transcription and translation rate can be assessed by determining ratios of extracellular luciferase to cellular activity. We recommend that the optimal harvest and collection times be assessed in pilot studies. For instance, extensive cell death results in reduced reporter production, and in the case of the apoptosis sensor used here, best results were seen when the cultures were assayed 24-32 hours after initiation of apoptosis. The assay is highly reproducible following transient transfection, and can also be used in cell lines stably transfected with the reporter if desired. Both transfected and endogenous protease activities are easily detected with this system. The transfer of a reporter enzyme across cell membranes constitutes an unexpected assay principle that adds a flexible, broadly applicable approach to current cell-based multi-color and multienzyme assays.
Applications of the protease sensor to study β-actin cleavage, apoptosis and autophagy
Cleavage of Actin-dNGLUC by HtrA3 and 4 suggests that members of the HtrA family of heat shock proteases, which are known to have significant functions in protein folding and apoptosis, may have the general property of cleaving actin in a manner that eliminates its ability to form insoluble fibers. Recently, a proteomic approach based on mass spectroscopic identification of cleavage products was undertaken to identify HTRA2 substrates . Major cleavage products included β-actin and tubulin alpha/beta and it was suggested that HTRA2 regulates apoptosis at the level of the cytoskeleton . Although β-actin has been reported as a substrate for a number of caspases, including caspase 3 , we have not observed release of dNGLUC from Actin-dNGLUC in response to caspase 3, 8 or 9, suggesting either that cleavage did not occur, or that it did not impair the ability of β-actin to anchor dNGLUC in the cell. In contrast to observations on cell-free extracts, cleavage of β-actin by caspases has not been detected in intact cells .
In a functional screen using the caspase sensor, we have identified the TBC family member TBC1D10A as an inducer of DEVDG-mediated cleavage. The TBC family of proteins exhibit GAP activity towards small GTPases of the Rab family . TBC1D10A has recently been identified as a GAP for Rab27A, suggesting a role in melanocyte transport and secretion . In addition, TBC1D10A binds to a complex of EBP50 with Ezrin and ARF6-GTP to regulate microvillus structure . Based on these data, TBC1D10A has been proposed as a regulator of protein trafficking in cells. Recently, a genome-wide screen for cell death effectors identified another family member, TBC1D10C, as an inducer of apoptosis . In agreement with this observation, our findings confirm a role for TBC1D10A as an effector of protein cleavage.
Autophagy is an essential cellular process for the degradation of proteins and organelles that has been associated with neurogenerative diseases, cancer and infection . Although autophagy is currently widely investigated, the systematic identification of molecular events in autophagy has been hampered by the lack of suitable assays. Current assays to study autophagy measure the accumulation of autophagic vacuoles by staining with fluorescent dyes such as monodansylcadaverine , or the sequestration of radioactive sugars or enzymes such as lactate dehydrogenase . However, these assays are difficult to quantify due to the presence of background levels of autophagic vacuoles or non-specific staining. Recently, immuno-blotting of hMAP1LC3 cleavage products, and GFPhMAP1LC3 translocation to autophagosomes  have been proposed as specific assays for autophagy. However, since the cleavage product of hMAP1LC3 is itself degraded by autophagy, interpretation of these assays requires additional controls . The assay presented here is a simple, easily implemented, quantitative assay that measures induction of autophagy without destruction of the cell being studied. As such, we anticipate it will be useful to many investigators in their studies of this enigmatic process.
It has been estimated that the human genome contains more than 500 proteases , most of which are poorly characterized. The luciferase secretion assay described here can be used to identify protease regulatory pathways as well as protease targets. The actions of nongenomic proteases, such as the HIV or HCV proteases or Anthrax lethal factor can be easily assessed by inserting the appropriate peptide target sequence in an actin-peptide-dNGLUC reporter construct.
The finding that Gaussia luciferase is capable of exiting the cell by a non-conventional secretion pathway is unusual in itself, and provides a tool to explore aspects of non-conventional secretion. Regulated non-conventional secretion of an enzymatic reporter has not been previously demonstrated to our knowledge, and affords several advantages over existing methods for analysis of intracellular cleavage events.
Of particular interest is the process of autophagy. Autophagy is a highly regulated process that appears to provide additional energy to cells under conditions of starvation. Autophagy has been suggested to play roles in the prevention and progression of cancers . The precise role that autophagy plays in these settings is not well understood, and high interest is currently directed toward understanding the contribution of autophagy to tumor growth. Large-scale screening approaches to identify regulators of autophagy to date have not been reported, possibly due to the absence of suitable screening assays. Analysis of autophagy is presently based on qualitative ultramorphological analyses, immunoblotting, or translocation of GFPLC3. Such assays can be non-quantitative, laborious and subject to multiple confounding factors . The analysis system described here facilitates insight into the regulation of autophagy and enables large scale shRNA knockdown and expression screening approaches.
Materials and methods
A GLUC sequence optimized for expression in both Escherichia coli and Homo sapiens was synthesized by tandem DNA oligonucleotide annealing and sub-cloned into pEAK12. During this process, the carboxy-terminal amino acid sequence LYK was added. Human β-actin was amplified from Origene Trueclone™ (AB1024H03) and inserted into the HindIII and NotI sites in pEAK12 using primers 5'-GACAAGCTTATGGATGATGATATCGCC-3' and 5'-GACGCGGCCGCTTAGAATTCGAAGCATTTGCGGTG-3'. dNGLUC was amplified by PCR using primers 5'-GACGAATTCATGCTAGCCAAGCCCACCG-3' and 5'-GGCTACTCTAGGGCACCTGTCCCGCC-3' and sub-cloned into pEAK12-βActin by digestion with EcoRI and NotI. A DEVDG(2)-Flag sequence was inserted at EcoRI as an adapter with the sequences 5'-AATTGGACGAGGTGGACGGCGACGAGGTGGACGGCGACTACAAGGACGA CGACGACAAGGAATTCGC-3' and 5'-GGCCGCGAATTCCTTGTCGTCGTCGTCCTTGTAGTCGCCGTCCACCTCGTC GCCGTCCACCTCGTCC-3' to generate pEAK12-Actin-DEVDG2flag-dNGLUC (DEVDG2F). Similarly, Actin-flagDEVDG2-dNGLUC (FDEVDG2) was constructed by inserting the Flag sequence before the DEVDG2 motif. A mutant Actin-DEVAG2flag-dNGLUC (DEVAG2F) construct was inserted with the same strategy. Actin-DEVDG3-dNGLUC (DEVDG3) was generated by introduction of three adjacent DEVDG sites. The Actin-LC3-dNGLUC construct was generated by PCR of hMAP1LC3 (Origene Trueclone AB2841G10) using primers 5'-GACGAATTCATGCCGTCGGAGAAGAC-3' and 5'-GACGCGGCCGCTTAGGATCCCACTGACAATTTCATCCC-3' and sub-cloned into the EcoRI and NotI site of pMOWSdSV. dNGLUC was amplified by PCR and inserted into the BamHI and NotI site of pMOWSdSV-LC3. The LC3-dNGLUC fusion was transferred by EcoRI and NotI digestion of pEAK12-β-actin to generate pEAK12-Actin-LC3-dNGLUC. GFP-dNGLUC was constructed by subcloning of dNGLUC into pEAK12-GFP using the EcoRI and NotI restriction sites. To generate Golgin67-DsRed, Golgin67 (Origene Trueclone AB1045_E08) was amplified by PCR and subcloned into pEAK12-GFP using HindIII and NotI restriction sites. DsRedExpress1 (Clontech, Mountain View, CA, USA) was amplified by PCR and sub-cloned in frame using EcoRI and NotI restriction sites. Expression vectors for caspase 8 and caspase 9 have been previously described . shRNA vectors for knockdown of human ATG4B (#TRCN0000073801), AKT1 (#TRCN0000010174) and vector control pLKO1 were obtained from Sigma (St. Louis, MO, USA).
293ET cells were cultured in DMEM (supplemented with 10% calf serum plus iron, 0.25 μg/ml gentamycin and 50 μM β-mercaptoethanol) and transfected using calcium phosphate precipitation as described elsewhere . The Origene Trueclone™ cDNA library consisting of approximately 12.000 human expression cDNAs arrayed in 96-well plates were transfected by TransFectin (BioRad, Hercules, CA, USA) along with a GFP expression construct in 293ET cells and screened for morphological changes by fluorescence microscopy (RK and BS, unpublished). Clones displaying signs of cell death were selected for transfection with Actin-DEVDG2F-dNGLUC. Supernatants were harvested after 24-32 h for luciferase analysis. Inhibitors of non-conventional secretion (7 μM Monensin, 10 μg/ml Brefeldin A, 5 μg/ml MG132; all from Sigma) were added 24 h after transfection and medium was collected over a 4 h time period.
Generation of stable 293ET cell line
Actin-dN and Actin-LC3-dN were subcloned into pMOWS  and co-transfected in 293ET cells with expression plasmids for VSV-G and retroviral gag-pol. The medium was changed after 24 h and virus supernatant was harvested and filtered through 0.45 μm filters 48 h after transfection. Untransfected 293ET cells were incubated with retroviral supernatant supplemented with 8 μg/ml polybrene; 48 h later, transduced 293ET cells were selected with puromycin at a concentration of 0.3 μg/ml.
A polyclonal antibody was raised in rabbit against dNGLUC (Proteintech Group Inc, Chicago, IL, USA). For western blotting, cells were lysed in 1% NP40 lysis buffer (20 mM Tris pH7.4, 150 mM NaCl, 1 mM EDTA, 1 mM ZnCl2, 1 mM MgCl2, 10% Glycerol) and resolved by SDS-PAGE. Proteins were blotted onto nitrocellulose (BioRad) and immune-stained with antibodies against dNGLUC, GFP (Covance, Princeton, NJ, USA), and Flag M2 (Sigma).
293ET cells were grown on coverslips and transfected by calcium phosphate precipitations as described. After 24 h, cells were fixed in 4% paraformaldehyde and mounted in aqueous mounting agent (Polysciences, Warrington, PA, USA). Images were obtained using confocal microscopy (BioRad Radiance 2000) and are a flat projection of z stacks taken throughout the plane of the transfected cell analyzed by LSM Image software (Carl Zeiss).
Luciferase and alkaline phosphatase assay
GLUC activity was determined using the Renilla Luciferase kit (Promega, Madison, WI, USA). To avoid harvesting luciferase activity from detached cells, supernatants were spun at 14,000 rpm for 5 minutes. Unless otherwise indicated, 10 μl of supernatant from a 12-well plate (total volume 1 ml) was diluted 1:10 in 100 μl 1 × Renilla lysis buffer and 10 μl of this mixture was added to 100 μl of Renilla substrate prior to analysis in a TopCount luminescence plate reader (Perkin Elmer, Waltham, MA, USA). For 96-well plates, 20 μl of SN was mixed with 20 μl of 2 × Renilla lysis buffer and 50 μl of Renilla substrate was added prior to analysis in the TopCount luminescence plate reader. Cells were lysed in 50 μl of 1 × Renilla lysis buffer and 25 μl of cell lysate was added to 50 μl of Renilla substrate. Secreted alkaline phosphatase was determined using the Phospha-Light™ secreted alkaline phosphatase reporter assay system (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Briefly, 50 μl of SN was mixed with 150 μl 1 × dilution buffer and incubated at 70°C for 20 minutes. Diluted SN (50 μl) was mixed with 50 μl of assay buffer and 50 μl of substrate solution before assaying in the TopCount luminescence plate reader.
fluorescence resonance energy transfer
GTPase activating protein
green fluorescent protein
Gaussia princeps luciferase
secreted alkaline phosphatase
small hairpin RNA
RK was supported by the Deutsche Forschungsgemeinschaft, Ke904/2-1. We thank Naifang Lu and Cathleen Tausch for experimental assistance, Tara Thurber for help with high-throughput screening, Vesko Tomov for helpful discussions, Soon-Young Na for critical evaluation of the manuscript, and Alan Huett and Ramnik Xavier for the GFP-LC3 construct and for critical evaluation of the manuscript.
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