Inositol 1,4,5-trisphosphate receptor type 1 is a substrate for caspase-3 and is cleaved during apoptosis in a caspase-3-dependent manner

The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), an IP(3)-gated Ca(2+) channel located on intracellular Ca(2+) stores, modulates intracellular Ca(2+) signaling. During apoptosis of the human T-cell line, Jurkat cells, as induced by staurosporine or Fas ligation, IP(3)R type 1 (IP(3)R1) was found to be cleaved. IP(3)R1 degradation during apoptosis was inhibited by pretreatment of Jurkat cells with the caspase-3 (-like protease) inhibitor, Ac-DEVD-CHO, and the caspases inhibitor, z-VAD-CH(2)DCB but not by the caspase-1 (-like protease) inhibitor, Ac-YVAD-CHO, suggesting that IP(3)R1 was cleaved by a caspase-3 (-like) protease. The recombinant caspase-3 cleaved IP(3)R1 in vitro to produce a fragmentation pattern consistent with that seen in Jurkat cells undergoing apoptosis. N-terminal amino acid sequencing revealed that the major cleavage site is (1888)DEVD*(1892)R (mouse IP(3)R1), which involves consensus sequence for caspase-3 cleavage (DEVD). To determine whether IP(3)R1 is cleaved by caspase-3 or is proteolyzed in its absence by other caspases, we examined the cleavage of IP(3)R1 during apoptosis in the MCF-7 breast carcinoma cell line, which has genetically lost caspase-3. Tumor necrosis factor-alpha- or staurosporine-induced apoptosis in caspase-3-deficient MCF-7 cells failed to demonstrate cleavage of IP(3)R1. In contrast, MCF-7/Casp-3 cells stably expressing caspase-3 showed IP(3)R1 degradation upon apoptotic stimuli. Therefore IP(3)R1 is a newly identified caspase-3 substrate, and caspase-3 is essential for the cleavage of IP(3)R1 during apoptosis. This cleavage resulted in a decrease in the channel activity as IP(3)R1 was digested, indicating that caspase-3 inactivates IP(3)R1 channel functions.     

Leader protein of foot-and-mouth disease virus is required for cleavage of the p220 component of the cap-binding protein complex.

Suppression of host protein synthesis in cells infected by poliovirus and certain other picornaviruses involves inactivation of the cap-binding protein complex. Inactivation of this complex has been correlated with the proteolytic cleavage of p220, a component of the cap-binding protein complex. Since picornaviral RNA is not capped, it continues to be translated as the cap-binding protein complex is inactivated. The cleavage of p220 can be induced to occur in vitro, catalyzed by extracts from infected cells or by reticulocyte lysates translating viral RNA. Expression of polioviral protease 2A is sufficient to induce p220 cleavage, and the presence in 2A of an 18-amino-acid sequence representing a putative cysteine protease active site correlates with the ability of different picornaviruses to induce p220 cleavage. Foot-and-mouth disease virus (FMDV) infection induces complete cleavage of p220, yet the FMDV genome codes for a 2A protein of only 16 amino acids, which does not include the putative cysteine protease active site. Using cDNA plasmids encoding various regions of the FMDV genome, we have determined that the leader protein is required to initiate p220 cleavage. This is the first report of a function for the leader protein, other than that of autocatalytic cleavage from the FMDV polyprotein.     

Enteroviral protease 2A directly cleaves dystrophin and is inhibited by a dystrophin-based substrate analogue

Enteroviruses such as Coxsackievirus B3 can cause dilated cardiomyopathy through unknown pathological mechanism(s). Dystrophin is a large extrasarcomeric cytoskeletal protein whose genetic deficiency causes hereditary dilated cardiomyopathy. In addition, we have recently shown that dystrophin is proteolytically cleaved by the Coxsackievirus protease 2A leading to functional impairment and morphological disruption. However, the mechanism of dystrophin cleavage and the exact cleavage site remained to be identified. Antibody epitope mapping of endogenous dystrophin indicated protease 2A-mediated cleavage at the site in the hinge 3 region predicted by a neural network algorithm (human, amino acid 2434; mouse, amino acid 2427). Using site-directed mutagenesis, peptide sequencing, and fluorescence resonance energy transfer assays with recombinant dystrophin, we demonstrate that this putative site in mouse and human dystrophin is a direct substrate for the Coxsackieviral protease 2A both in vitro and in vivo. The substrate analogue protease inhibitor z-LSTT-fmk was designed based on the dystrophin sequence that interacts with the protease 2A and was found to have an IC(50) of 550 nM in vitro. Dystrophin is the first cellular substrate of the enteroviral protease 2A that was identified using by a bioinformatic approach and for which the cleavage site was molecularly mapped within living cells.     

A fluorogenic substrate as quantitative in vivo reporter to determine protein expression and folding of tobacco etch virus protease in Escherichia coli

Quantitative and folding reporters are adequate tools to optimize recombinant protein expression in various host organisms, including Escherichia coli. To determine the yield of soluble active protease from the tobacco etch virus (TEV), we developed a single-molecule assay based on the fluorogenic substrate ANA-QS-MCA. This substrate consists of a 10 amino acid peptide (ENLYFQSGTK) containing the proteolytic cleavage sequence of the TEV protease. The peptide works as a linker N-terminally tagged with a fluorescent donor group (7-Methoxycoumarin-4-yl)acetyl (MCA) and C-terminally tagged with the acceptor group 5-Amino-2-nitrobenzoic acid (ANA). Fluorescence can be observed after specific cleavage of the substrate at the Gln-Ser bond by active TEV protease. Purified His-tagged TEV protease was used for in vitro analysis. Through determination of proteolytic activity in living E. coli cells and through application of Confocal Laser-Scanning-Microscopy we demonstrate that the peptide is well suited to in vivo expression analysis. This provides an effective tool to monitor the accumulation of active recombinant TEV protease in crude extracts and intact cells.     

Processing of the human transferrin receptor at distinct positions within the stalk region by neutrophil elastase and cathepsin G

The ectodomain of the human transferrin receptor (TfR) is released as soluble TfR into the blood by cleavage within a stalk. The major cleavage site is located C-terminally of Arg-100; alternative cleavage sites are also present. Since the cleavage process is still unclear, we looked for proteases involved in TfR ectodomain release. In the supernatant of U937 histiocytic cells we detected alternatively cleaved TfR (at Glu-110). In membrane fractions of these cells we identified two distinct proteolytic activities responsible for TfR cleavage within the stalk at either Val-108 or Lys-95. Both activities could be inhibited by serine protease inhibitors, but not by inhibitors of any other class of proteases. Protein purification yielded a 28 kDa protein that generated the Val-108 terminus. The protease activity could be ascribed to neutrophil elastase according to the substrate specificity determined by amino acid substitution analysis of synthetic peptides, an inhibitor profile, the size of the protease and the use of specific antibodies. The results of analogous experiments suggest that the second activity is represented by another serine protease, cathepsin G. Thus, membrane-associated forms of neutrophil elastase and cathepsin G may be involved in alternative TfR shedding in U937 cells.     

Linkers for improved cleavage of fusion proteins with an engineered alpha-lytic protease.

Addition of an N-terminal fusion partner can greatly aid the expression and purification of a recombinant protein in Escherichia coli. We investigated two genetically engineered proteases designed to remove the fusion partner after the protein of interest has been expressed. Recombinant human insulin-like growth factor-II (hIGF-II) has been produced from E. coli-derived fusion proteins using a novel enzymatic cleavage system that uses a mutant of alpha-lytic protease. Initially, two potential fusion protein linkers were designed, Pro-Ala-Pro-His (PAPH) and Pro-Ala-Pro-Met (PAPM), and were tested as substrates in the form of synthetic dodecapeptides. Using mass spectrometry and reverse-phase HPLC, the position of cleavage was confirmed and the kinetics of synthetic peptide cleavage were examined. Use of the linkers in hIGF-II fusion proteins produced in E. coli was then evaluated. The fusion proteins constructed consist of the first 11 amino acids of porcine growth hormone linked N-terminally to hIGF-II by six amino acids that include the dipeptide Val-Asn followed by a variable tetrapeptide protease cleavage motif. Mass spectrometry and N-terminal sequencing confirmed that proteolytic cleavage of the fusion proteins had occurred at the predicted sites. Using the fusion proteins as substrates, the cleavage of the rationally designed motifs by the alpha-lytic protease mutant was compared. The fusion protein containing the motif PAPM had a k(cat)/K(M) ratio indicating a 1.6-fold preference over the PAPH fusion protein for cleavage by this enzyme. Furthermore, when hIGF-II fusion proteins containing the designed cleavable linkers were processed with the engineered alpha-lytic protease, they gave greatly improved yields of native hIGF-II compared to an analogous fusion protein cleaved by H64A subtilisin. Comparison of the peptide and protein cleavage studies shows that the efficient proteolysis of the cleavage motifs is an inherent property of the designed sequences and is not determined by secondary or tertiary structure in the fusion proteins.     

Proteolytic activity assayed by subcellular localization switching of a substrate

An approach to assay proteolytic activity in vivo by altering the subcellular localization of a labelled substrate was demonstrated. The assay included a protein shuttling between different cellular compartments and a site-specific recombinant protease. The shuttle protein used was the human immunodeficiency virus type 1 (HIV-1) Rev protein tandemly fused to the enhanced green fluorescent protein (EGFP) and the red fluorescent protein (RFP), while the protease was the site-specific protease VP24 from the herpes simplex virus type 1 (HSV-1). The fluorescent proteins in the Rev fusion protein were separated by a cleavage site specific for the VP24 protease. When co-expressed in COS-7 cells proteolysis was observed by fluorescence microscopy as a shift from a predominantly cytoplasmic localization of the fusion protein RevEGFP to a nuclear localization while the RFP part of the fusion protein remained in the cytoplasm. The cleavage of the fusion protein by VP24 was confirmed by Western blot analysis. The activity of VP24, when tagged N-terminally by the Myc-epitope, was found to be comparable to VP24. These results demonstrates that the activity and localization of a recombinantly expressed protease can be assessed by protease-mediated cleavage of fusion proteins containing a specific protease cleavage site.     

The development-associated cleavage of lens connexin 45.6 by caspase-3-like protease is regulated by casein kinase II-mediated phosphorylation.

Gap junctions are important in maintaining lens transparency and metabolic homeostasis. In this paper, we report that the gap junction-forming protein, connexin (Cx) 45.6, was specifically truncated during lens development and that the majority of the truncated fragments were located in the differentiated lens fibers. When isolated lens membranes were treated by caspase-3, the truncated fragments of Cx45.6 were reproduced, and this truncation occurred at the COOH terminus of Cx45.6. Moreover, when primary lens cells were treated with apoptosis-inducing reagents, Cx45.6 was cleaved similarly as the in vitro treatment by caspase-3, and this cleavage was blocked by a caspase-3 inhibitor. These results suggest that caspase-3 is responsible for the development-associated cleavage of Cx45.6. The cleavage site of Cx45.6 was identified between amino acid residues Glu(367) and Gly(368). We have shown previously that Ser(363) is an in vivo phosphorylated site by casein kinase II, and this specific phosphorylation leads to a rapid turnover of Cx45.6. Interestingly, we found here that when Ser(363) was phosphorylated by casein kinase II, the cleavage of Cx45.6 catalyzed by caspase-3 was inhibited. This study, for the first time, demonstrates that a connexin can be a direct target of an apoptotic protease and that cleavage by caspase-3-like protease leads to the development-associated truncation of a lens connexin. Finally, caspase-3-mediated cleavage can be regulated by casein kinase II-mediated phosphorylation, suggesting that Cx45.6 turnover and specific cleavage by caspase-3-like protease is alternatively modulated.     

Identification of candidate substrates for ectodomain shedding by the metalloprotease-disintegrin ADAM8

ADAM proteases are type I transmembrane proteins with extracellular metalloprotease domains. As for most ADAM family members, ADAM8 (CD156a, MS2) is involved in ectodomain shedding of membrane proteins and is linked to inflammation and neurodegeneration. To identify potential substrates released under these pathologic conditions, we screened 10-mer peptides representing amino acid sequences from extracellular domains of various membrane proteins using the ProteaseSpot system. A soluble ADAM8 protease containing a pro- and metalloprotease domain was expressed in E. coli and purified as active protease owing to autocatalytic prodomain removal. From 34 peptides tested in the peptide cleavage assay, significant cleavage by soluble ADAM8 was observed for 14 peptides representing membrane proteins with functions in inflammation and neurodegeneration, among them the beta-amyloid precursor protein (APP). The in vivo relevance of the ProteaseSpot method was confirmed by cleavage of full-length APP with ADAM8 in human embryonic kidney 293 cells expressing tagged APP. ADAM8 cleaved APP with similar efficiency as ADAM10, whereas the inactive ADAM8 mutant did not. Exchanging amino acids at defined positions in the cleavage sequence of myelin basic protein (MBP) revealed sequence criteria for ADAM8 cleavage. Taken together, the results allowed us to identify novel candidate substrates that could be cleaved by ADAM8 in vivo under pathologic conditions.     

Cleavage of eukaryotic translation initiation factor 4GII within foot-and-mouth disease virus-infected cells: identification of the L-protease cleavage site in vitro

Foot-and-mouth disease virus (FMDV) induces a very rapid inhibition of host cell protein synthesis within infected cells. This is accompanied by the cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI). The cleavage of the related protein eIF4GII has now been analyzed. Within FMDV-infected cells, cleavage of eIF4GI and eIF4GII occurs with similar kinetics. Cleavage of eIF4GII is induced in cells and in cell extracts by the FMDV leader protease (L(pro)) alone, generating cleavage products similar to those induced by enterovirus and rhinovirus 2A protease (2A(pro)). By the use of a fusion protein containing residues 445 to 744 of human eIF4GII, it was demonstrated that the FMDV L(pro) specifically cleaves this protein between residues G700 and S701, immediately adjacent to the site (V699/G700) cleaved by rhinovirus 2A(pro) in vitro. The G700/S701 cleavage site does not correspond, by amino acid sequence alignment, to that cleaved in eIF4GI by the FMDV L(pro) in vitro. Knowledge of the cleavage sites and the three-dimensional structures of the FMDV L(pro) and rhinovirus 2A(pro) enabled mutant forms of the eIF4GII sequence to be generated that are differentially resistant to either one of these proteases. These results confirmed the specificity of each protease and showed that the mutant forms of the fusion protein substrate retained their correct sensitivity to other proteases.     

Comparison of substrate specificities against the fusion glycoprotein of virulent Newcastle disease virus between a chick embryo fibroblast processing protease and mammalian subtilisin-like proteases

The fusion (F) protein precursor of virulent Newcastle disease virus (NDV) strains has two pairs of basic amino acids at the cleavage site, and its intracellular cleavage activation occurs in a variety of cells; therefore, the viruses cause systemic infections in poultry. To explore the protease responsible for the cleavage in the natural host, we examined detailed substrate specificity of the enzyme in chick embryo fibroblasts (CEF) using a panel of the F protein mutants at the cleavage site expressed by vaccinia virus vectors, and compared the specificity with those of mammalian subtilisin-like proteases such as furin, PC6 and PACE4 which are candidates for F protein processing enzymes. It was demonstrated in CEF cells that Arg residues at the -4, -2 and -1 positions upstream of the cleavage site were essential, and that at the -5 position was required for maximal cleavage. Phe at the +1 position was also important for efficient cleavage. On the other hand, furin and PC6 expressed by vaccinia virus vectors showed cleavage specificities against the F protein mutants consistent with that shown by the processing enzyme of CEF cells, but PACE4 hardly cleaved the F proteins including the wild type. These results indicate that the proteolytic processing enzymes of poultry for virulent NDV F proteins could be furin and/or PC6 but not PACE4. The significance of individual contribution of the three amino acids at the -5, -2 and +1 positions to cleavability was discussed in relation to the evolution of virulent and avirulent NDV strains.     

Type 1 inositol-1,4,5-trisphosphate receptor is a late substrate of caspases during apoptosis

Apoptosis is characterized by the proteolytic cleavage of hundreds of proteins. One of them, the type 1 inositol-1,4,5-trisphosphate receptor (IP(3) R-1), a multimeric receptor located on the endoplasmic reticulum (ER) membrane that is critical to calcium homeostasis, was reported to be cleaved during staurosporine (STS) induced-apoptosis in Jurkat cells. Because the reported cleavage site separates the IP(3) binding site from the channel moiety, its cleavage would shut down a critical signaling pathway that is common to several cellular processes. Here we show that IP(3) R-1 is not cleaved in 293 cells treated with STS, TNFα, Trail, or ultra-violet (UV) irradiation. Further, it is not cleaved in Hela or Jurkat cells induced to undergo apoptosis with Trail, TNFα, or UV. In accordance with previous reports, we demonstrate that it is cleaved in a Jurkat cell line treated with STS. However its cleavage occurs only after poly(ADP-ribose) polymerase (PARP), which cleavage is a hallmark of apoptosis, and p23, a poor caspase-7 substrate, are completely cleaved, suggesting that IP(3) R-1 is a relatively late substrate of caspases. Nevertheless, the receptor is fully accessible to proteolysis in cellulo by ectopically overexpressed caspase-7 or by the tobacco etch virus (TEV) protease. Finally, using recombinant caspase-3 and microsomal fractions enriched in IP(3) R-1, we show that the receptor is a poor caspase-3 substrate. Consequently, we conclude that IP(3) R-1 is not a key death substrate.     

Synthesis and processing of the transmembrane envelope protein of equine infectious anemia virus.

The transmembrane (TM) envelope protein of lentiviruses, including equine infectious anemia virus (EIAV), is significantly larger than that of other retroviruses and may extend in the C-terminal direction 100 to 200 amino acids beyond the TM domain. This size difference suggests a lentivirus-specific function for the long C-terminal extension. We have investigated the synthesis and processing of the EIAV TM protein by immune precipitation and immunoblotting experiments, by using several envelope-specific peptide antisera. We show that the TM protein in EIAV particles is cleaved by proteolysis to an N-terminal glycosylated 32- to 35-kilodalton (kDa) segment and a C-terminal nonglycosylated 20-kDa segment. The 20-kDa fragment was isolated from virus fractionated by high-pressure liquid chromatography, and its N-terminal amino acid sequence was determined for 13 residues. Together with the known nucleotide sequence, this fixes the cleavage site at a His-Leu bond located 240 amino acids from the N terminus of the TM protein. Since the 32- to 35-kDa fragment and the 20-kDa fragment are not detectable in infected cells, we assume that cleavage occurs in the virus particle and that the viral protease may be responsible. We have also found that some cells producing a tissue-culture-adapted strain of EIAV synthesize a truncated envelope precursor polyprotein. The point of truncation differs slightly in the two cases we have observed but lies just downstream from the membrane-spanning domain, close to the cleavage point described above. In one case, virus producing the truncated envelope protein appeared to be much more infectious than virus producing the full-size protein, suggesting that host cell factors can select for virus on the basis of the C-terminal domain of the TM protein.     

The HtrA (DegP) protein, essential for Escherichia coli survival at high temperatures, is an endopeptidase.

As a preliminary step in the understanding of the function of the Escherichia coli HtrA (DegP) protein, which is indispensable for bacterial survival only at elevated temperatures, the protein was purified and partially characterized. The HtrA protein was purified from cells carrying the htrA gene cloned into a multicopy plasmid, resulting in its overproduction. The sequence of the 13 N-terminal amino acids of the purified HtrA protein was determined and was identical to the one predicted for the mature HtrA protein by the DNA sequence of the cloned gene. Moreover, the N-terminal sequence showed that the 48-kilodalton HtrA protein is derived by cleavage of the first 26 amino acids of the pre-HtrA precursor polypeptide and that the point of cleavage follows a typical target sequence recognized by the leader peptidase enzyme. The HtrA protein was shown to be a specific endopeptidase which was inhibited by diisopropylfluorophosphate, suggesting that HtrA is a serine protease.     

Proteolysis of bacteriophage phi X174 prohead protein gpB by a protease located in the Escherichia coli outer membrane.

The gene B protein (gpB) of bacteriophage phi X174 is required for prohead assembly and is removed from prohead during phage maturation. Protease activity was observed in isolated prohead which specifically cleaved gpB. Cleavage of gpB produced two fragments that had apparent molecular weights of 12,300 and 3,700 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal sequencing of the fragments confirmed that they resulted from the cleavage of gpB and identified the cleavage site as an Arg-Arg at amino acids 76 to 77 of the 120-amino-acid protein. gpB-specific protease activity was observed in both phi X174-infected and uninfected Escherichia coli extracts. This protease activity was localized to the outer-membrane fraction of uninfected cells. Protease activities present in the outer membrane and in isolated prohead produced identical fragments and had the same protease inhibition profile. The gpB-specific activity in uninfected cells was induced by growth at 42 degrees C and was inhibited by the protease inhibitors, 1,10-phenanthroline, EDTA, and N-ethylmaleimide.     

The use of recombinant fusion proteases in the affinity purification of recombinant proteins

In the affinity purification of recombinant fusion proteins, the rate-limiting step is usually the efficient proteolytic cleavage and removal of the affinity tail and the protease from the purified recombinant protein. We have developed a rapid, convenient, and efficient method of affinity purification that can overcome this limitation. In one example of the method, the protease 3C from a picornavirus (3Cpro), which cleaves specific sequences containing a minimum of 6-7 amino acids, has been expressed as a fusion with glutathione S-transferase. The resultant recombinant "fusion protease" cleaves fusion proteins bearing (from the amino-terminus) the same affinity tail as the fusion protease, a 3Cpro cleavage recognition site, and the recombinant protein of interest. The recombinant protein is purified in a single chromatographic step, which removes both the affinity tail and the fusion protease. The advantages over existing methods include much improved specificity of proteolytic cleavage, complete removal of the protease and the affinity tail in one step, and the option of adding any desired amount of fusion protease to ensure efficient cleavage. The potential flexibility of the method is shown by the use of various affinity tails and alternative fusion proteases.     

Retinoic acid-induced upregulation of the metalloendopeptidase nardilysin is accelerated by co-expression of the brain-specific protein p42(IP4) (centaurin α1; ADAP1) in neuroblastoma cells

The mainly neuronally expressed protein p42^IP4 (centaurin α1; ADAP1), which interacts with the metalloendopeptidase nardilysin (NRD) was found to be localized in neuritic plaques in Alzheimer disease (AD) brains. NRD was shown to enhance the cleavage of the amyloid precursor protein (APP) by α-secretases, thereby increasing the release of neuroprotective sAPPα. We here investigated in vitro the biochemical interaction of p42^IP4 and NRD and studied the physiological interaction in SH-SY5Y cells. NRD is a member of the M16 family of metalloendopeptidases. Some members of this M16 family act bi-functionally, as protease and as non-enzymatic scaffold protein. Here, we show that p42^IP4 enhances the enzymatic activity of NRD 3-4 times. However, p42^IP4 is not a substrate for NRD. Furthermore, we report that differentiation of SH-SY5Y cells by stimulation with 10 μM retinoic acid (RA) results in upregulation of NRD protein levels, with a 6-fold rise after 15 days. NRD is expressed in the neurites of RA-stimulated SH-SY5Y cells, and localized in vesicular structures. Since p42^IP4 is not expressed in untreated SH-SY5Y cells, we could use this cell system as a model to find out, whether there is a functional interaction. Interestingly, SH-SY5Y cells, which we stably transfected with GFP-tagged-p42^IP4 showed an enhanced NRD protein expression already at an earlier time point after RA stimulation.