Perforin is activated by a proteolytic cleavage during biosynthesis which reveals a phospholipid-binding C2 domain.

Perforin is a secreted protein synthesized by activated cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. It is a key component of the lytic machinery of these cells, being able to insert into the plasma membrane of targeted cells, forming a pore which leads to their destruction. Here we analyse the synthesis, processing and intracellular transport of perforin in the NK cell line YT. Perforin is synthesized as a 70 kDa inactive precursor which is cleaved at the C-terminus to yield a 60 kDa active form. This proteolytic cleavage occurs in an acidic compartment and can be inhibited by incubation of the cells in ammonium chloride, concanamycin A, leupeptin and E-64. The increased lytic activity of the cleaved form can be demonstrated by killing assays in which cleavage of the pro-piece is inhibited. Epitope mapping reveals that cleavage of the pro-piece occurs at the boundary of a C2 domain, which we show is able to bind phospholipid membranes in a calcium-dependent manner. We propose that removal of the pro-piece, which contains a bulky glycan, allows the C2 domain to interact with phospholipid membranes and initiate perforin pore formation.     

Identification of subtilisin, Epr and Vpr as enzymes that produce CSF, an extracellular signalling peptide of Bacillus subtilis

Cell-cell communication regulates many important processes in bacteria. Gram-positive bacteria use peptide signals for communication, such as the Phr pentapeptides of Bacillus subtilis. The Phr pentapeptides are secreted with a pro domain that is cleaved to produce an active signalling peptide. To identify the protease(s) involved in production of the mature Phr signalling peptides, we developed assays for detecting cleavage of one of the B. subtilis Phr pentapeptides, CSF, from the proCSF precursor. Using both a cellular and a mass spectrometric approach, we determined that a sigma-H-regulated, secreted, serine protease(s) cleaved proCSF to CSF. Mutants lacking the three proteases that fit these criteria, subtilisin, Epr and Vpr, had a defect in CSF production. Purified subtilisin and Vpr were shown to be capable of processing proCSF as well as at least one other Phr peptide produced by B. subtilis, PhrA, but they were not able to process the PhrE signalling peptide of B. subtilis, indicating that there are probably other unidentified proteases involved in Phr peptide production. Subtilisin, Epr and Vpr are members of the subtilisin family of proteases that are widespread in bacteria, suggesting that many bacterial species may be capable of producing Phr signalling peptides.     

Efficient double-stranded DNA cleavage by artificial zinc-finger nucleases composed of one zinc-finger protein and a single-chain FokI dimer

Zinc-finger–FokI nucleases (ZFNs) are useful for manipulating genomic DNA, but two ZFNs are required to cleave one site of double-stranded DNA (dsDNA), which limits the choice of targets. To refine ZFN technology, we constructed artificial zinc-finger nucleases containing an artificial zinc-finger protein (AZP) and a single-chain FokI dimer with nine different peptide linkers between two FokI molecules (designated AZP–scFokI). DNA cleavage assays revealed that the AZP–scFokI variant possessing the longest peptide linker cleaved dsDNA with equal or greater reactivity than the corresponding AZP–FokI dimer. The DNA cleavage pattern of AZP–scFokI suggests that the enhanced dsDNA cleavage was due to increased formation of FokI dimer in AZP–scFokI. Furthermore, we demonstrated that AZP–scFokI site-specifically cleaved its target DNA due to the AZP moiety discriminating one base pair difference. Thus, a single AZP–scFokI molecule is able to cleave dsDNA efficiently and site-specifically, and enhances the usefulness of the ZFN approach.     

Feline sarcoma virus-coded polyprotein: enzymatic cleavage by a type C virus-coded structural protein.

A purified 15,000-molecular-weight (Mr) Prague strain Rous sarcoma virus gag gene-coded structural protein, p15, was shown to enzymatically cleave the previously described 130,000 Mr feline sarcoma virus-coded polyprotein, Pr130. Cleavage products included proteins ranging in molecular weight from 12,000 to 110,000. The specificity of this cleavage reactivity was indicated by the fact that, under similar conditions, neither purified type C viral structural proteins nor nonviral proteins such as bovine serum albumin were cleaved to significant extents. Moreover, feline leukemia virus Pr65gag was efficiently cleaved, resulting in the generations of proteins of 30,000 (p30), 15,000 (p15), 12,000 (p12), and 10,000 (p10) Mr. Using enzymatically (p15) treated feline sarcoma virus Pr130 as starting material, we were able to purify a major 72,000 Mr cleavage product and to show it to contain the previously described feline sarcoma virus-coded nonstructural component.     

Characterization of a human angiotensinogen cleaved in its reactive center loop by a proteolytic activity from Chinese hamster ovary cells

Angiotensinogen, the renin (E.C. 3.4.23.15) substrate, belongs to the serpins superfamily and has been classified as a noninhibitory serpin. Using mass spectroscopy, angiotensinogen purified from Chinese hamster ovary cell supernatant shows a broad spectrum. The absence of protease inhibitors throughout the purification leads to an angiotensinogen cleaved within the reactive center loop. This cleavage does not affect the Ang I generation because kinetic parameters are similar to the values of the full-length angiotensinogen. Although cleavage is complete, the cleaved angiotensinogen migrates after deglycosylation on SDS-polyacrylamide gel electrophoresis as a doublet differing by 4 kDa. To test whether the circulating angiotensinogen is cleaved in the reactive center loop, it was purified from a pool of human plasma and was shown to be uncleaved. Its migration was obviously slower than of cleaved angiotensinogen but also consisted of two bands pointing to a so far unexplained residual heterogeneity. We then compared the heat-induced polymerization of full-length- and reactive center loop-cleaved angiotensinogens. Both monomers were able to aggregate, revealing a particular behavior of angiotensinogen distinct from that of reactive center loop-cleaved serpins. Lacking the three-dimensional structure of angiotensinogen, we propose and discuss a structural model of the serpin fold within the renin substrate.     

Elucidating the Specificity Determinants of the AtxE2 Lasso Peptide Isopeptidase

Lasso peptide isopeptidase is an enzyme that specifically hydrolyzes the isopeptide bond of lasso peptides, rendering these peptides linear. To carry out a detailed structure-activity analysis of the lasso peptide isopeptidase AtxE2 from Asticcacaulis excentricus, we solved NMR structures of its substrates astexin-2 and astexin-3. Using in vitro enzyme assays, we show that the C-terminal tail portion of these peptides is dispensable with regards to isopeptidase activity. A collection of astexin-2 and astexin-3 variants with alanine substitutions at each position within the ring and the loop was constructed, and we showed that all of these peptides except for one were cleaved by the isopeptidase. Thus, much like the lasso peptide biosynthetic enzymes, lasso peptide isopeptidase has broad substrate specificity. Quantitative analysis of the cleavage reactions indicated that alanine substitutions in loop positions of these peptides led to reduced cleavage, suggesting that the loop is serving as a recognition element for the isopeptidase.     

A faster migrating variant masquerades as NICD when performing in vitro gamma-secretase assays with bacterially expressed Notch substrates

Intramembrane proteolysis is a new and rapidly growing field. In vitro assays utilizing recombinant substrates for gamma-secretase, an intramembrane-cleaving enzyme, are critically important in order to characterize the biochemical properties of this unusual enzyme. Several recombinant Notch proteins of varying length are commonly used as in vitro substrates for CHAPSO-solubilized gamma-secretase. Here we report that several recombinant Notch constructs undergo limited or no proteolysis in vitro. Instead, upon incubation with or without gamma-secretase, variants of the intact protein migrate during SDS-PAGE at the location expected for the gamma-secretase specific cleavage products. In addition, we show that addition of aspartyl- and gamma-secretase specific protease inhibitors are able to retard the formation of these variants independent of gamma-secretase, which could lead to the erroneous conclusion that Notch cleavage by solubilized gamma-secretase was achieved in vitro even when no proteolysis occurred. In contrast, substrates produced in mammalian or insect cells are cleaved efficiently in vitro. These observations suggest that in vitro studies reliant on recombinant, bacterially produced Notch TMD should be performed with the inclusion of additional controls able to differentiate between actual cleavage and this potential artifact.     

Cleaved SLPI, a novel biomarker of chymase activity

Secretory leukocyte protease inhibitor (SLPI) is a protease inhibitor of the whey acidic protein-like family inhibiting chymase, chymotrypsin, elastase, proteinase 3, cathepsin G and tryptase. Performing in vitro enzymatic assays using both Western blotting and liquid chromatography/mass spectrometry techniques we showed that, of the proteases known to interact with SLPI, only chymase could uniquely cleave this protein. The peptides of the cleaved SLPI (cSLPI) remain coupled due to the disulfide bonds in the molecule but under reducing conditions the cleavage can be observed as peptide products. Subsequent ex vivo studies confirmed the presence of SLPI in human saliva and its susceptibility to cleavage by chymase. Furthermore, inhibitors of chymase activity are able to inhibit this cleavage. Human saliva from both normal and allergic individuals was analyzed for levels of cSLPI and a correlation between the level of cSLPI and the extent of allergic symptoms was observed, suggesting the application of cSLPI as a biomarker of chymase activity in humans.     

Down-regulation of Delta by proteolytic processing

Notch signaling regulates cell fate decisions during development through local cell interactions. Signaling is triggered by the interaction of the Notch receptor with its transmembrane ligands expressed on adjacent cells. Recent studies suggest that Delta is cleaved to release an extracellular fragment, DlEC, by a mechanism that involves the activity of the metalloprotease Kuzbanian; however, the functional significance of that cleavage remains controversial. Using independent functional assays in vitro and in vivo, we examined the biological activity of purified soluble Delta forms and conclude that Delta cleavage is an important down-regulating event in Notch signaling. The data support a model whereby Delta inactivation is essential for providing the critical ligand/receptor expression differential between neighboring cells in order to distinguish the signaling versus the receiving partner.     

Foot-and-mouth disease virus 3C protease induces cleavage of translation initiation factors eIF4A and eIF4G within infected cells

Infection of cells by foot-and-mouth disease virus (FMDV) results in the rapid inhibition of host cell protein synthesis. This process is accompanied by the early cleavage of the translation initiation factor eIF4G, a component of the cap-binding complex eIF4F. This cleavage is mediated by the leader (L) protease. Subsequently, as the virus proteins accumulate, secondary cleavages of eIF4G occur. Furthermore, eIF4A (46 kDa), a second component of eIF4F, is also cleaved in these later stages of the infection cycle. The 33-kDa cleavage product of eIF4A has lost a fragment from its N terminus. Transient-expression assays demonstrated that eIF4A was not cleaved in the presence of FMDV L or with the poliovirus 2A protease (which also mediates eIF4G cleavage) but was cleaved when the FMDV 3C protease was expressed. The FMDV 3C protease was also shown in such assays to induce cleavage of eIF4G, resulting in the production of cleavage products different from those generated by the L protease. Consistent with these results, within cells infected with a mutant FMDV lacking the L protease or within cells containing an FMDV replicon lacking L-P1 coding sequences it was again shown that eIF4A and eIF4G were cleaved.     

Biological,immunological and biochemical characterization of cleaved prolactin generated by lactating mammary gland

We have previously shown that rat prolactin is proteolytically cleaved in its loop by peripheral tissues of the rat. Of the tissues examined to date, lactating mammary gland exhibits the highest prolactin-cleaving activity. The objective of this study was to characterize cleaved prolactin, biologically, immunologically and chemically. By modifying an established analytical method, we were able to generate large (μg) amounts of cleaved rat prolactin from cell fractions of rat mammary gland which could then be assayed for biological and immunological activity relative to intact hormone. The cleaved product showed no significant difference relative to the intact rat prolactin when assayed for its ability to compete with ¹²⁵I-labelled ovine prolactin for the prolactin receptor and for its ability to stimulate the proliferation of rat Nb2 lymphoma cells. Cleaved rat prolactin, however, did show a 50–60% reduction in activity relative to intact rat prolactin when assayed by radioimmunoassay. Using Edman degradation and partial amino acid analysis, we determined that the second N-terminus of the cleaved rat prolactin begins at amino acid 149. The divergence of biological and immunological activity produced by proteolytic cleavage in the loop of rat prolactin suggests that biological and immunological sites differ in location. The possible physiological implications of a cleaved rat prolactin molecule generated by target tissue with maintained biological activity and reduced immunological activity are discussed.     

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.     

Factor VIIa/tissue factor generates a form of factor V with unchanged specific activity, resistance to activation by thrombin, and increased sensitivity to activated protein C

Factor VIIa, in complex with tissue factor (TF), is the serine protease responsible for initiating the clotting cascade. This enzyme complex (TF/VIIa) has extremely restricted substrate specificity, recognizing only three previously known macromolecular substrates (serine protease zymogens, factors VII, IX, and X). In this study, we found that TF/VIIa was able to cleave multiple peptide bonds in the coagulation cofactor, factor V. SDS-PAGE analysis and sequencing indicated the factor V was cleaved at Arg679, Arg709, Arg1018, and Arg1192, resulting in a molecule with a truncated heavy chain and an extended light chain. This product (FVTF/VIIa) had essentially unchanged activity in clotting assays when compared to the starting material. TF reconstituted into phosphatidylcholine vesicles was ineffective as a cofactor for the factor VIIa cleavage of factor V. However, incorporation of phosphatidylethanolamine in the vesicles had little effect over the presence of 20% phosphatidylserine. FVTF/VIIa was as sensitive to inactivation by activated protein C (APC) as thrombin activated factor V as measured in clotting assays or by the appearance of the expected heavy chain cleavage products. The FVTF/VIIa could be further cleaved by thrombin to release the normal light chain, albeit at a significantly slower rate than native factor V, to yield a fully functional product. These studies thus reveal an additional substrate for the TF/VIIa complex. They also indicate a new potential regulatory pathway of the coagulation cascade, i.e., the production of a form of factor V that can be destroyed by APC without the requirement for full activation of the cofactor precursor.     

Proteolysis of HIP during apoptosis occurs within a region similar to the BID loop

BID is an essential component of many apoptotic pathways. Cytosolic proteases cleave BID within an extended loop region, generating an active truncated fragment which synergizes with BAX and BAK to induce release of apoptogenic factors from mitochondria. To determine whether other proteins are cleaved in a similar manner as BID, we performed a database search for proteins which possess sequence similarity with the BID loop region. One of the proteins identified was the Hsc70-interacting protein (HIP). We analyzed the cleavage pattern of HIP using two known activators of BID: granzyme B and caspase-8. In in vitro cleavage assays using recombinant proteins, human and rat HIP were cleaved by granzyme B. Furthermore, the granzyme B-mediated cleavage site was mapped to the BID loop-like region of HIP by site-directed mutagenesis. This region was also the target for caspase-8-mediated cleavage in rat HIP. However, human HIP was not proteolyzed by caspase-8, which probably reflects sequence differences between human and rat HIP proteins at the P₁′ position of the caspase-8 recognition sequence. To determine whether HIP is cleaved during apoptosis, human Jurkat T cells were exposed to granzyme B and perforin. The results of these studies suggest that granzyme B-mediated loss of HIP expression occurs in vivo, and in a coordinate fashion with loss of BID, pro-caspase-8 and pro-caspase-3. These data implicate the Hsp70 co-chaperone HIP in the proteolytic cascade of some apoptotic pathways.     

Advances in cytochemical methods for detection of apoptosis.

In an earlier article from this laboratory, the current methods developed to detect apoptosis in cells and tissues were highlighted, along with the challenges in their interpretation. Recent discoveries concerning the underlying biochemical mechanisms of apoptotic effector pathways have made possible further assays that allow a more direct measure of the activation of the apoptotic machinery in cells. This article summarizes some of these newer methods and extends the interpretation of the more classical assays of apoptosis in a defined cell system. We present data in KB and PC3 cell model culture systems induced to undergo apoptosis by the plant toxin ricin. Using a modified in situ nick translation assay (ISNT) with either Bodipy or BUdR labeling, we confirm that most cells showing altered nuclear morphology do not show reactivity with this assay until very late in the apoptotic process. We also show that only a minority of cells label with fluorescent annexin V during apoptosis but that apoptotic cells continue to internalize material from the cell surface through endocytosis after becoming reactive with annexin V. In addition, we describe the utility of a prototype of new assays for caspase substrate cleavage products, the detection of cleaved cytokeratin 18. It is these newer cleavage product assays that perhaps hold the greatest promise for specific detection of apoptosis in cells either in cell culture or in intact tissues. (J Histochem Cytochem 49:821-832, 2001)     

Perpetuating the homing endonuclease life cycle: identification of mutations that modulate and change I-TevI cleavage preference

Homing endonucleases are sequence-tolerant DNA endonucleases that act as mobile genetic elements. The ability of homing endonucleases to cleave substrates with multiple nucleotide substitutions suggests a high degree of adaptability in that changing or modulating cleavage preference would require relatively few amino acid substitutions. Here, using directed evolution experiments with the GIY-YIG homing endonuclease I-TevI that targets the thymidylate synthase gene of phage T4, we readily isolated variants that dramatically broadened I-TevI cleavage preference, as well as variants that fine-tuned cleavage preference. By combining substitutions, we observed an ∼10 000-fold improvement in cleavage on some substrates not cleaved by the wild-type enzyme, correlating with a decrease in readout of information content at the cleavage site. Strikingly, we were able to change the cleavage preference of I-TevI to that of the isoschizomer I-BmoI which targets a different cleavage site in the thymidylate synthase gene, recapitulating the evolution of cleavage preference in this family of homing endonucleases. Our results define a strategy to isolate GIY-YIG nuclease domains with distinct cleavage preferences, and provide insight into how homing endonucleases may escape a dead-end life cycle in a population of saturated target sites by promoting transposition to different target sites.     

A Novel Two-Tag System for Monitoring Transport and Cleavage through the Classical Secretory Pathway – Adaptation to HIV Envelope Processing

The classical secretory pathway is essential for the transport of a host of proteins to the cell surface and/or extracellular matrix. While the pathway is well-established, many factors still remain to be elucidated. One of the most relevant biological processes that occur during transport involves the cleavage of pro-proteins by enzymes residing in the endoplasmic reticulum/Golgi/TransGolgi Network compartment. Teasing out the requirements involved in the classical secretory pathway and cleavage during transport would shed new light into mis-regulation leading to disease. Current methodologies fail to link transport and cleavage at the single cell level. Here, we describe a cell-based assay that relies on an engineered protein scaffold that can discriminate between transport to the cell surface, in the absence or presence of cleavage. Our novel two-tag system works in a robust and quantitative manner and distinguishes between cleaved and non-cleaved events based on cell surface expression of one or two epitope tags, respectively. Here, we have used the HIV-1 envelope as a substrate, which is cleaved during transport, as proof of principle. Importantly, this assay can be easily coupled to existing siRNA-based screens to identify novel regulators and effectors involved in transport and/or cleavage of cell surface proteins. In addition, unlike other in vivo based assays, the assay described here can also be easily adapted to drug discovery purposes.     

A proteomic approach to the identification of molecular targets in subsequent apoptosis of HEL cells after diosgenin‐induced megakaryocytic differentiation

Diosgenin is a plant steroid which is able to induce megakaryocytic differentiation of human erythroleukemia (HEL) cells followed by apoptosis at a later stage. Apoptosis markers and phospho‐kinases involved during the subsequent apoptosis of megakaryocytes after diosgenin‐induced differentiation in these cells were detected using a proteomic approach. In mature megakaryocytes undergoing apoptosis, we observed increased expression of intrinsic apoptosis markers such as Bax/Bcl‐2 ratio and cleaved caspase‐9 as well as extrinsic apoptosis markers including cell death receptors and cleaved caspase‐8. Furthermore, we demonstrated the link between both apoptotic pathways by Bid cleavage and confirmed the executive phase of apoptosis by caspase‐3 cleavage. For the first time, we examined kinase activation and showed that kinases including Src, Tor, Akt, CREB, RSK and Chk2 may be implicated in signalling of subsequent apoptosis of mature megakaryocytes after diosgenin‐induced differentiation of HEL cells. J. Cell. Biochem. 107: 785–796, 2009. © 2009 Wiley‐Liss, Inc.