Calpain-1 Cleaves and Activates Caspase-7

Caspase-7 is an executioner caspase that plays a key role in apoptosis, cancer, and a number of neurodegenerative diseases. The mechanism of caspase-7 activation by granzyme B and caspase-3 has been well characterized. However, whether other proteases such as calpains activate or inactivate caspase-7 is not known. Here, we present that recombinant caspase-7 is directly cleaved by calpain-1 within the large subunit of caspase-7 to produce two novel products, large subunit p18 and p17. This new form of caspase-7 has a 6-fold increase in V_max when compared with the previously characterized p20/p12 form. Zymography revealed that the smaller caspase-7 product (p17) is 18-fold more active than either the caspase-3-cleaved product (p20) or the larger calpain-1 product of caspase-7 (p18). Mass spectrometry and site-directed mutagenesis identified the calpain cleavage sites within the caspase-7 large subunit at amino acid 36 and 45/47. These proteolysis events occur in vivo as indicated by the accumulation of caspase-7 p18 and p17 subunits in cortical neurons undergoing Ca²⁺ dysregulation. Further, cleavage at amino acid 45/47 of caspase-7 by calpain results in a reduction in nuclear localization when compared with the caspase-3 cleavage product of caspase-7 (p20). Our studies suggest the calpain-activated form of caspase-7 has unique enzymatic activity, localization, and binding affinity when compared with the caspase-activated form.Apoptosis is a well-defined cellular destruction pathway that primarily utilizes a family of cysteine proteases, the caspases (1, 2). This cell death program can be initiated by cell death receptor activation (extrinsic pathway) or a variety of drugs or cellular stresses (intrinsic pathway) leading to activation of apical caspase-8, -9, and/or -10 (1, 3, 4). These initiator caspases in turn directly activate the executioner caspases, caspase-3 and -7, which through proteolysis of defined substrates are responsible for the dismantling of the cell and subsequent death (3, 4). Granzyme B, released by cytotoxic T lymphocytes to protect the host from pathogens and tumor cells, can also initiate this apoptotic cascade and therefore is considered an apical caspase mimic (5–7). All caspases, as well as granzyme B, preferentially cleave after aspartic acid residues, with many having well-defined consensus sequences, making substrate cleavage sites easy to predict and establish (3, 4, 7, 8).Caspases exist in a latent form prior to activation. Both the initiator and executioner caspases are synthesized as a single chain protein, which require proteolytic cleavage to become active. Procaspase-7 is expressed as a 303-amino acid residue polypeptide chain. The activation and regulation of executioner caspase-7 by caspases and granzyme B has been extensively studied. Caspase-7 requires cleavage by caspase-3 and caspase-8/-10 or granzyme B, for activation (6, 9). Current evidence suggests that caspase-3 initially cleaves off the first 23 amino acids (propeptide, 2 kDa), followed by caspase-8/-10 or granzyme B cleaving between the large (20 kDa) and small (12 kDa) subunit after amino acid 198 to activate the enzyme. The large subunit containing the catalytic His-237 and Cys-285 (caspase-1 numbering convention), and the small subunit are involved in the formation of the substrate-binding region. In vitro, granzyme B can also activate caspase-7 independently of caspase-3, but this does not appear to occur in vivo (5, 6). Currently, there is no evidence that other classes of proteases play a role in activating or modulating caspase-7 activity.Changes in intracellular Ca²⁺ levels influence apoptosis in a number of cell types (10–13). Because in many of these apoptotic cell models the Ca²⁺-dependent cysteine proteases, calpains, are activated upstream of caspases (14–16), it is possible that calpains may activate and/or modulate caspase activity via direct cleavage. Studies directed at understanding calpains with respect to caspase activation are limited. Calpain-2 was shown to cleave procaspase-9, decreasing its activity (17). In the same study, calpain-2 treatment cleaved procaspase-7 to produce a single, novel fragment, but in this case the effect on enzymatic activity was not investigated (17). To improve our understanding of calpains and the role of calcium in cell death, we carried out studies directed at understanding how calpains activate or modulate caspase activity. We found that calpain treatment produced a large increase in caspase-7 activity. Calpain cleaves procaspase-7 to produce two large subunits of 18.5 and 17.2 kDa, the smaller of which has a robust increase in activity relative to the 20-kDa large subunit produced by caspase-3 cleavage of caspase-7. Both calpain cleavage sites in caspase-7 are identified using mass spectrometry. N-methyl-d-aspartate-induced Ca²⁺-dependent cell death in primary cortical neurons produced calpain-derived caspase-7 cleavage products in vivo. Lastly, the strictly cytosolic localization of the smaller calpain fragment confirms that a previously identified nuclear localization signal (18) is involved in caspase-7 cytosolic/nuclear distribution. Our data suggest that increases in Ca²⁺ leading to activation of calpains may significantly modulate caspase-7 activity and thus, apoptosis.     

In Vitro Characterization of a Recombinant Blh Protein from an Uncultured Marine Bacterium as a ��-Carotene 15,15��-Dioxygenase

Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved β-carotene at its central double bond (15,15′) to yield two molecules of all-trans-retinal. The molecular mass of the native purified enzyme was ∼64 kDa as a dimer of 32-kDa subunits. The K_m, k_cat, and k_cat/K_m values for β-carotene as substrate were 37 μm, 3.6 min⁻¹, and 97 mm⁻¹ min⁻¹, respectively. The enzyme exhibited the highest activity for β-carotene, followed by β-cryptoxanthin, β-apo-4′-carotenal, α-carotene, and γ-carotene in decreasing order, but not for β-apo-8′-carotenal, β-apo-12′-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C₃₅ seems to be essential for enzyme activity. The oxygen atom of retinal originated not from water but from molecular oxygen, suggesting that the enzyme was a β-carotene 15,15′-dioxygenase. Although the Blh protein and β-carotene 15,15′-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian β-carotene 15,15′-monooxygenases as assessed by their different properties, including DNA and amino acid sequences, molecular weight, form of association, reaction mechanism, kinetic properties, and substrate specificity. This is the first report of in vitro characterization of a bacterial β-carotene-cleaving enzyme.Vitamin A (retinol) is a fat-soluble vitamin and important for human health. In vivo, the cleavage of β-carotene to retinal is an important step of vitamin A synthesis. The cleavage can proceed via two different biochemical pathways (1, 2). The major pathway is a central cleavage catalyzed by mammalian β-carotene 15,15′-monooxygenases (EC 1.14.99.36). β-Carotene is cleaved by the enzyme symmetrically into two molecules of all-trans-retinal, and retinal is then converted to vitamin A in vivo (3–5). The second pathway is an eccentric cleavage that occurs at double bonds other than the central 15,15′-double bond of β-carotene to produce β-apo-carotenals with different chain lengths, which are catalyzed by carotenoid oxygenases from mammals, plants, and cyanobacteria (6). These β-apo-carotenals are degraded to one molecule of retinal, which is subsequently converted to vitamin A in vivo (2).β-Carotene 15,15′-monooxygenase was first isolated as a cytosolic enzyme by identifying the product of β-carotene cleavage as retinal (7). The characterization of the enzyme and the reaction pathway from β-carotene to retinal were also investigated (4, 8). The enzyme activity has been found in mammalian intestinal mucosa, jejunum enterocytes, liver, lung, kidney, and brain (5, 9, 10). Molecular cloning, expression, and characterization of β-carotene 15,15′-monooxygenase have been reported from various species, including chickens (11), fruit flies (12), humans (13), mice (14), and zebra fishes (15).Other proteins thought to convert β-carotene to retinal include bacterioopsin-related protein (Brp) and bacteriorhodopsin-related protein-like homolog protein (Blh) (16). Brp protein is expressed from the bop gene cluster, which encodes the structural protein bacterioopsin, consisting of at least three genes as follows: bop (bacterioopsin), brp (bacteriorhodopsin-related protein), and bat (bacterioopsin activator) (17). brp genes were reported in Haloarcula marismortui (18), Halobacterium sp. NRC-1 (19), Halobacterium halobium (17), Haloquadratum walsbyi, and Salinibacter ruber (20). Blh protein is expressed from the proteorhodopsin gene cluster, which contains proteorhodopsin, crtE (geranylgeranyl-diphosphate synthase), crtI (phytoene dehydrogenase), crtB (phytoene synthase), crtY (lycopene cyclase), idi (isopentenyl diphosphate isomerase), and blh gene (21). Sources of blh genes were previously reported in Halobacterium sp. NRC-1 (19), Haloarcula marismortui (18), Halobacterium salinarum (22), uncultured marine bacterium 66A03 (16), and uncultured marine bacterium HF10 49E08 (21). β-Carotene biosynthetic genes crtE, crtB, crtI, crtY, ispA, and idi encode the enzymes necessary for the synthesis of β-carotene from isopentenyl diphosphate, and the Idi, IspA, CrtE, CrtB, CrtI, and CrtY proteins have been characterized in vitro (23–28). Blh protein has been proposed to catalyze or regulate the conversion of β-carotene to retinal (29, 30), but there is no direct proof of the enzymatic activity.In this study, we used codon optimization to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli, and we performed a detailed biochemical and enzymological characterization of the expressed Blh protein. In addition, the properties of the enzyme were compared with those of mammalian β-carotene 15,15′-monooxygenases.     

Repeated Domains of Leptospira Immunoglobulin-like Proteins Interact with Elastin and Tropoelastin

Leptospira spp., the causative agents of leptospirosis, adhere to components of the extracellular matrix, a pivotal role for colonization of host tissues during infection. Previously, we and others have shown that Leptospira immunoglobulin-like proteins (Lig) of Leptospira spp. bind to fibronectin, laminin, collagen, and fibrinogen. In this study, we report that Leptospira can be immobilized by human tropoelastin (HTE) or elastin from different tissues, including lung, skin, and blood vessels, and that Lig proteins can bind to HTE or elastin. Moreover, both elastin and HTE bind to the same LigB immunoglobulin-like domains, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12 as demonstrated by enzyme-linked immunosorbent assay (ELISA) and competition ELISAs. The LigB immunoglobulin-like domain binds to the 17th to 27th exons of HTE (17–27HTE) as determined by ELISA (LigBCon4, K_D = 0.50 μm; LigBCen7′–8, K_D = 0.82 μm; LigBCen9, K_D = 1.54 μm; and LigBCen12, K_D = 0.73 μm). The interaction of LigBCon4 and 17–27HTE was further confirmed by steady state fluorescence spectroscopy (K_D = 0.49 μm) and ITC (K_D = 0.54 μm). Furthermore, the binding was enthalpy-driven and affected by environmental pH, indicating it is a charge-charge interaction. The binding affinity of LigBCon4D341N to 17–27HTE was 4.6-fold less than that of wild type LigBCon4. In summary, we show that Lig proteins of Leptospira spp. interact with elastin and HTE, and we conclude this interaction may contribute to Leptospira adhesion to host tissues during infection.Pathogenic Leptospira spp. are spirochetes that cause leptospirosis, a serious infectious disease of people and animals (1, 2). Weil syndrome, the severe form of leptospiral infection, leads to multiorgan damage, including liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, meningitis, abortion, and uveitis (3, 4). Furthermore, this disease is not only prevalent in many developing countries, it is reemerging in the United States (3). Although leptospirosis is a serious worldwide zoonotic disease, the pathogenic mechanisms of Leptospira infection remain enigmatic. Recent breakthroughs in applying genetic tools to Leptospira may facilitate studies on the molecular pathogenesis of leptospirosis (5–8).The attachment of pathogenic Leptospira spp. to host tissues is critical in the early phase of Leptospira infection. Leptospira spp. adhere to host tissues to overcome mechanical defense systems at tissue surfaces and to initiate colonization of specific tissues, such as the lung, kidney, and liver. Leptospira invade hosts tissues through mucous membranes or injured epidermis, coming in contact with subepithelial tissues. Here, certain bacterial outer surface proteins serve as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)² to mediate the binding of bacteria to different extracellular matrices (ECMs) of host cells (9). Several leptospiral MSCRAMMs have been identified (10–18), and we speculate that more will be identified in the near future.Lig proteins are distributed on the outer surface of pathogenic Leptospira, and the expression of Lig protein is only found in low passage strains (14, 16, 17), probably induced by environmental cues such as osmotic or temperature changes (19). Lig proteins can bind to fibrinogen and a variety of ECMs, including fibronectin (Fn), laminin, and collagen, thereby mediating adhesion to host cells (20–23). Lig proteins also constitute good vaccine candidates (24–26).Elastin is a component of ECM critical to tissue elasticity and resilience and is abundant in skin, lung, blood vessels, placenta, uterus, and other tissues (27–29). Tropoelastin is the soluble precursor of elastin (28). During the major phase of elastogenesis, multiple tropoelastin molecules associate through coacervation (30–32). Because of the abundance of elastin or tropoelastin on the surface of host cells, several bacterial MSCRAMMs use elastin and/or tropoelastin to mediate adhesion during the infection process (33–35).Because leptospiral infection is known to cause severe pulmonary hemorrhage (36, 37) and abortion (38), we hypothesize that some leptospiral MSCRAMMs may interact with elastin and/or tropoelastin in these elastin-rich tissues. This is the first report that Lig proteins of Leptospira interact with elastin and tropoelastin, and the interactions are mediated by several specific immunoglobulin-like domains of Lig proteins, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12, which bind to the 17th to 27th exons of human tropoelastin (HTE).     

Loop 2 Structure in Glycine and GABAA Receptors Plays a Key Role in Determining Ethanol Sensitivity

The present study tests the hypothesis that the structure of extracellular domain Loop 2 can markedly affect ethanol sensitivity in glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABA_ARs). To test this, we mutated Loop 2 in the α1 subunit of GlyRs and in the γ subunit of α1β2γ2GABA_ARs and measured the sensitivity of wild type and mutant receptors expressed in Xenopus oocytes to agonist, ethanol, and other agents using two-electrode voltage clamp. Replacing Loop 2 of α1GlyR subunits with Loop 2 from the δGABA_AR (δL2), but not the γGABA_AR subunit, reduced ethanol threshold and increased the degree of ethanol potentiation without altering general receptor function. Similarly, replacing Loop 2 of the γ subunit of GABA_ARs with δL2 shifted the ethanol threshold from 50 mm in WT to 1 mm in the GABA_A γ-δL2 mutant. These findings indicate that the structure of Loop 2 can profoundly affect ethanol sensitivity in GlyRs and GABA_ARs. The δL2 mutations did not affect GlyR or GABA_AR sensitivity, respectively, to Zn²⁺ or diazepam, which suggests that these δL2-induced changes in ethanol sensitivity do not extend to all allosteric modulators and may be specific for ethanol or ethanol-like agents. To explore molecular mechanisms underlying these results, we threaded the WT and δL2 GlyR sequences onto the x-ray structure of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC). In addition to being the first GlyR model threaded on GLIC, the juxtaposition of the two structures led to a possible mechanistic explanation for the effects of ethanol on GlyR-based on changes in Loop 2 structure.Alcohol abuse and dependence are significant problems in our society, with ∼14 million people in the United States being affected (1, 2). Alcohol causes over 100,000 deaths in the United States, and alcohol-related issues are estimated to cost nearly 200 billion dollars annually (2). To address this, considerable attention has focused on the development of medications to prevent and treat alcohol-related problems (3–5). The development of such medications would be aided by a clear understanding of the molecular structures on which ethanol acts and how these structures influence receptor sensitivity to ethanol.Ligand-gated ion channels (LGICs)² have received substantial attention as putative sites of ethanol action that cause its behavioral effects (6–12). Research in this area has focused on investigating the effects of ethanol on two large superfamilies of LGICs: 1) the Cys-loop superfamily of LGICs (13, 14), whose members include nicotinic acetylcholine, 5-hydroxytryptamine₃, γ-aminobutyric acid type A (GABA_A), γ-aminobutyric acid type C, and glycine receptors (GlyRs) (10, 11, 15–20) and 2) the glutamate superfamily, including N-methyl d-aspartate, α-amino-3-hydroxyisoxazolepropionic acid, and kainate receptors (21, 22). Recent studies have also begun investigating ethanol action in the ATP-gated P2X superfamily of LGICs (23–25).A series of studies that employed chimeric and mutagenic strategies combined with sulfhydryl-specific labeling identified key regions within Cys-loop receptors that appear to be initial targets for ethanol action that also can determine the sensitivity of the receptors to ethanol (7–12, 18, 19, 26–30). This work provides several lines of evidence that position 267 and possibly other sites in the transmembrane (TM) domain of GlyRs and homologous sites in GABA_ARs are targets for ethanol action and that mutations at these sites can influence ethanol sensitivity (8, 9, 26, 31).Growing evidence from GlyRs indicates that ethanol also acts on the extracellular domain. The initial findings came from studies demonstrating that α1GlyRs are more sensitive to ethanol than are α2GlyRs despite the high (∼78%) sequence homology between α1GlyRs and α2GlyRs (32). Further work found that an alanine to serine exchange at position 52 (A52S) in Loop 2 can eliminate the difference in ethanol sensitivity between α1GlyRs and α2GlyRs (18, 20, 33). These studies also demonstrated that mutations at position 52 in α1GlyRS and the homologous position 59 in α2GlyRs controlled the sensitivity of these receptors to a novel mechanistic ethanol antagonist (20). Collectively, these studies suggest that there are multiple sites of ethanol action in α1GlyRs, with one site located in the TM domain (e.g. position 267) and another in the extracellular domain (e.g. position 52).Subsequent studies revealed that the polarity of the residue at position 52 plays a key role in determining the sensitivity of GlyRs to ethanol (20). The findings with polarity in the extracellular domain contrast with the findings at position 267 in the TM domain, where molecular volume, but not polarity, significantly affected ethanol sensitivity (9). Taken together, these findings indicate that the physical-chemical parameters of residues at positions in the extracellular and TM domains that modulate ethanol effects and/or initiate ethanol action in GlyRs are not uniform. Thus, knowledge regarding the physical-chemical properties that control agonist and ethanol sensitivity is key for understanding the relationship between the structure and the actions of ethanol in LGICs (19, 31, 34–40).GlyRs and GABA_ARs, which differ significantly in their sensitivities to ethanol, offer a potential method for identifying the structures that control ethanol sensitivity. For example, α1GlyRs do not reliably respond to ethanol concentrations less than 10 mm (32, 33, 41). Similarly, γ subunit-containing GABA_ARs (e.g. α1β2γ2), the most predominantly expressed GABA_ARs in the central nervous system, are insensitive to ethanol concentrations less than 50 mm (42, 43). In contrast, δ subunit-containing GABA_ARs (e.g. α4β3δ) have been shown to be sensitive to ethanol concentrations as low as 1–3 mm (44–51). Sequence alignment of α1GlyR, γGABA_AR, and δGABA_AR revealed differences between the Loop 2 regions of these receptor subunits. Since prior studies found that mutations of Loop 2 residues can affect ethanol sensitivity (19, 20, 39), the non-conserved residues in Loop 2 of GlyR and GABA_AR subunits could provide the physical-chemical and structural bases underlying the differences in ethanol sensitivity between these receptors.The present study tested the hypothesis that the structure of Loop 2 can markedly affect the ethanol sensitivity of GlyRs and GABA_ARs. To accomplish this, we performed multiple mutations that replaced the Loop 2 region of the α1 subunit in α1GlyRs and the Loop 2 region of the γ subunit of α1β2γ2 GABA_ARs with corresponding non-conserved residues from the δ subunit of GABA_AR and tested the sensitivity of these receptors to ethanol. As predicted, replacing Loop 2 of WT α1GlyRs with the homologous residues from the δGABA_AR subunit (δL2), but not the γGABA_AR subunit (γL2), markedly increased the sensitivity of the receptor to ethanol. Similarly, replacing the non-conserved residues of the γ subunit of α1β2γ2 GABA_ARs with δL2 also markedly increased ethanol sensitivity of GABA_ARs. These findings support the hypothesis and suggest that Loop 2 may play a role in controlling ethanol sensitivity across the Cys-loop superfamily of receptors. The findings also provide the basis for suggesting structure-function relationships in a new molecular model of the GlyR based on the bacterial Gloeobacter violaceus pentameric LGIC homologue (GLIC).     

Broad Coverage Identification of Multiple Proteolytic Cleavage Site Sequences in Complex High Molecular Weight Proteins Using Quantitative Proteomics as a Complement to Edman Sequencing

Proteolytic processing modifies the pleiotropic functions of many large, complex, and modular proteins and can generate cleavage products with new biological activity. The identification of exact proteolytic cleavage sites in the extracellular matrix laminins, fibronectin, and other extracellular matrix proteins is not only important for understanding protein turnover but is needed for the identification of new bioactive cleavage products. Several such products have recently been recognized that are suggested to play important cellular regulatory roles in processes, including angiogenesis. However, identifying multiple cleavage sites in extracellular matrix proteins and other large proteins is challenging as N-terminal Edman sequencing of multiple and often closely spaced cleavage fragments on SDS-PAGE gels is difficult, thus limiting throughput and coverage. We developed a new liquid chromatography-mass spectrometry approach we call amino-terminal oriented mass spectrometry of substrates (ATOMS) for the N-terminal identification of protein cleavage fragments in solution. ATOMS utilizes efficient and low cost dimethylation isotopic labeling of original N-terminal and proteolytically generated N termini of protein cleavage fragments followed by quantitative tandem mass spectrometry analysis. Being a peptide-centric approach, ATOMS is not dependent on the SDS-PAGE resolution limits for protein fragments of similar mass. We demonstrate that ATOMS reliably identifies multiple proteolytic sites per reaction in complex proteins. Fifty-five neutrophil elastase cleavage sites were identified in laminin-1 and fibronectin-1 with 34 more identified by matrix metalloproteinase cleavage. Hence, our degradomics approach offers a complimentary alternative to Edman sequencing with broad applicability in identifying N termini such as cleavage sites in complex high molecular weight extracellular matrix proteins after in vitro cleavage assays. ATOMS can therefore be useful in identifying new cleavage products of extracellular matrix proteins cleaved by proteases in pathology for bioactivity screening.Recently, considerable efforts have been deployed to develop high throughput proteomic screens to identify protease substrates in complex biological samples (1–8). Validation of substrates identified by these approaches or identification of cleavage sites by in vitro incubation of candidate substrates with the protease of interest is generally performed by SDS-PAGE analysis and Edman degradation and sequencing. However, the complexity of large modular proteins renders Edman sequencing of proteolytic fragments difficult to apply because each of the numerous proteolytic fragments should be analyzed separately, and high coverage of cleavage sites is rarely attained (9). Cleavage site identification after protein degradation is also very difficult for small peptide products less than 4 kDa. Consequently, the precise cleavage sites in complex extracellular matrix proteins such as laminin and fibronectin by important tissue and inflammatory cell proteases such as the matrix metalloproteinases (MMPs)1 and neutrophil elastase are mostly unknown.These limitations of Edman sequencing are problematic in the study of tissue remodeling and proteolysis in pathology. Neutrophil elastase and several MMPs such as MMP2, MMP8, and MMP9 play key roles in inflammation (10, 11), tissue healing (12, 13), and carcinogenesis (14, 15) and are well known for degrading extracellular matrix proteins (16). More recently, signaling functions for MMPs are increasingly recognized as one of their most important roles by the precise processing of cytokines or their binding proteins (17). In addition, several important examples are now known of cryptic binding sites being exposed after precise protein cleavage or new proteins termed neoproteins (18) being released upon limited cleavage of extracellular matrix proteins and having completely different functions compared with their parent molecule, including several with importance in angiogenesis (19–25). Many such sites or neoproteins are generated by inflammatory proteases or proteases of the coagulation and fibrinolysis systems (24, 25), and this is a burgeoning field of discovery that is often hampered by difficulties in their N-terminal sequencing.In light of this limitation, we developed, validated, and used a new method for targeted and simultaneous N-terminal sequencing of one or a small number of protein N termini or cleavage products we call amino-terminal oriented mass spectrometry of substrates (ATOMS). We applied ATOMS for the analysis of cleavage sites generated in laminin-1 and fibronectin-1 by neutrophil elastase and neutrophil and tissue MMPs. Laminin-1 (LM-111), a trimeric glycoprotein composed of the α1, β1, and γ1 chains, is ubiquitously expressed in epithelium and endothelium. Proteolytic processing of laminins greatly affects cellular behavior and is also implicated in cancer cell migration (20, 26–29). Another important extracellular matrix protein is plasma fibronectin (also known as fibronectin isoform 1) and its cellular isoforms, which are homodimers linked by a disulfide bridge at the C terminus (30) that are important for cell adhesion and intracellular signaling (31–34). Fibronectin is susceptible to proteolysis (35, 36), which affects its biological functions (37–39). However, the cleavage sites within these two molecules by inflammatory MMPs and neutrophil elastase are largely unknown. Here we identified a total of 55 neutrophil elastase cleavage sites in LM-111 and fibronectin-1 and 34 MMP cleavage sites, demonstrating the capacity of ATOMS to identify multiple N-terminal sequences in solution. ATOMS also outperformed N-terminal Edman sequencing with 50% more cleavage sites identified by ATOMS, representing a significant advance in N-terminal sequencing technology. The utility of the method is broadly applicable for the analysis of multiple cleavages in other very large molecules and so offers great potential to accurately identify and rapidly sequence multiple cryptic bioactive protein fragments liberated following proteolytic processing.     

��-Defensins in Enteric Innate Immunity: FUNCTIONAL PANETH CELL ��-DEFENSINS IN MOUSE COLONIC LUMEN*

Paneth cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric α-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric α-defensins of Paneth cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative α-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as α-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1–4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth cell α-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts.Antimicrobial peptides (AMPs)² are released by epithelial cells onto mucosal surfaces as effectors of innate immunity (1–5). In mammals, most AMPs derive from two major families, the cathelicidins and defensins (6). The defensins comprise the α-, β-, and θ-defensin subfamilies, which are defined by the presence of six cysteine residues paired in characteristic tridisulfide arrays (7). α-Defensins are highly abundant in two primary cell lineages: phagocytic leukocytes, primarily neutrophils, of myeloid origin and Paneth cells, which are secretory epithelial cells located at the base of the crypts of Lieberkühn in the small intestine (8–10). Neutrophil α-defensins are stored in azurophilic granules and contribute to non-oxidative microbial cell killing in phagolysosomes (11, 12), except in mice whose neutrophils lack defensins (13). In the small bowel, α-defensins and other host defense proteins (14–18) are released apically as components of Paneth cell secretory granules in response to cholinergic stimulation and after exposure to bacterial antigens (19). Therefore, the release of Paneth cell products into the crypt lumen is inferred to protect mitotically active crypt cells from colonization by potential pathogens and confer protection against enteric infection (7, 20, 21).Under normal, homeostatic conditions, Paneth cells are not found outside the small bowel, although they may appear ectopically in response to local inflammation throughout the gastrointestinal tract (22, 23). Paneth cell numbers increase progressively throughout the small intestine, occurring at highest numbers in the distal ileum (24). Mouse Paneth cells express numerous α-defensin isoforms, termed cryptdins (Crps) (25), that have broad spectrum antimicrobial activities (6, 26). Collectively, α-defensins constitute approximately seventy percent of the bactericidal peptide activity in mouse Paneth cell secretions (19), selectively killing bacteria by membrane-disruptive mechanisms (27–30). The role of Paneth cell α-defensins in gastrointestinal mucosal immunity is evident from studies of mice transgenic for human enteric α-defensin-5, HD-5, which are immune to infection by orally administered Salmonella enterica sv. typhimurium (S. typhimurium) (31).The biosynthesis of mature, bactericidal α-defensins from their inactive precursors requires activation by lineage-specific proteolytic convertases. In mouse Paneth cells, inactive ∼8.4-kDa Crp precursors are processed intracellularly into microbicidal ∼4-kDa Crps by specific cleavage events mediated by matrix metalloproteinase-7 (MMP-7) (32, 33). MMP-7 null mice exhibit increased susceptibility to systemic S. typhimurium infection and decreased clearance of orally administered non-invasive Escherichia coli (19, 32). Although the α-defensin proregions are sensitive to proteolysis, the mature, disulfide-stabilized peptides resist digestion by their converting enzymes in vitro, whether the convertase is MMP-7 (32), trypsin (34), or neutrophil serine proteinases (35). Because α-defensins resist proteolysis in vitro, we hypothesized that Paneth cell α-defensins resist degradation and remain in a functional state in the large bowel, a complex, hostile environment containing varied proteases of both host and microbial origin.Here, we report on the isolation and characterization of a population of enteric α-defensins from the mouse colonic lumen. Full-length and N-terminally truncated Paneth cell α-defensins were identified and are abundant in the distal large bowel lumen.     

Deletion of the Chloride Transporter Slc26a7 Causes Distal Renal Tubular Acidosis and Impairs Gastric Acid Secretion

SLC26A7 (human)/Slc26a7 (mouse) is a recently identified chloride-base exchanger and/or chloride transporter that is expressed on the basolateral membrane of acid-secreting cells in the renal outer medullary collecting duct (OMCD) and in gastric parietal cells. Here, we show that mice with genetic deletion of Slc26a7 expression develop distal renal tubular acidosis, as manifested by metabolic acidosis and alkaline urine pH. In the kidney, basolateral Cl⁻/HCO₃⁻ exchange activity in acid-secreting intercalated cells in the OMCD was significantly decreased in hypertonic medium (a normal milieu for the medulla) but was reduced only mildly in isotonic medium. Changing from a hypertonic to isotonic medium (relative hypotonicity) decreased the membrane abundance of Slc26a7 in kidney cells in vivo and in vitro. In the stomach, stimulated acid secretion was significantly impaired in isolated gastric mucosa and in the intact organ. We propose that SLC26A7 dysfunction should be investigated as a potential cause of unexplained distal renal tubular acidosis or decreased gastric acid secretion in humans.The collecting duct segment of the distal kidney nephron plays a major role in systemic acid base homeostasis by acid secretion and bicarbonate absorption. The acid secretion occurs via H⁺-ATPase and H-K-ATPase into the lumen and bicarbonate is absorbed via basolateral Cl⁻/HCO₃⁻ exchangers (1–4). The tubules, which are located within the outer medullary region of the kidney collecting duct (OMCD),² have the highest rate of acid secretion among the distal tubule segments and are therefore essential to the maintenance of acid base balance (2).The gastric parietal cell is the site of generation of acid and bicarbonate through the action of cytosolic carbonic anhydrase II (5, 6). The intracellular acid is secreted into the lumen via gastric H-K-ATPase, which works in conjunction with a chloride channel and a K⁺ recycling pathway (7–10). The intracellular bicarbonate is transported to the blood via basolateral Cl⁻/HCO₃⁻ exchangers (11–14).SLC26 (human)/Slc26 (mouse) isoforms are members of a conserved family of anion transporters that display tissue-specific patterns of expression in epithelial cells (15–24). Several SLC26 members can function as chloride/bicarbonate exchangers. These include SLC26A3 (DRA), SLC26A4 (pendrin), SLC26A6 (PAT1 or CFEX), SLC26A7, and SLC26A9 (25–31). SLC26A7 and SLC26A9 can also function as chloride channels (32–34).SLC26A7/Slc26a7 is predominantly expressed in the kidney and stomach (28, 29). In the kidney, Slc26a7 co-localizes with AE1, a well-known Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of (acid-secreting) A-intercalated cells in OMCD cells (29, 35, 36) (supplemental Fig. 1). In the stomach, Slc26a7 co-localizes with AE2, a major Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of acid secreting parietal cells (28). To address the physiological function of Slc26a7 in the intact mouse, we have generated Slc26a7 ko mice. We report here that Slc26a7 ko mice exhibit distal renal tubular acidosis and impaired gastric acidification in the absence of morphological abnormalities in kidney or stomach.     

Structure of the c14 Rotor Ring of the Proton Translocating Chloroplast ATP Synthase

The structure of the membrane integral rotor ring of the proton translocating F₁F₀ ATP synthase from spinach chloroplasts was determined to 3.8 Å resolution by x-ray crystallography. The rotor ring consists of 14 identical protomers that are symmetrically arranged around a central pore. Comparisons with the c₁₁ rotor ring of the sodium translocating ATPase from Ilyobacter tartaricus show that the conserved carboxylates involved in proton or sodium transport, respectively, are 10.6–10.8 Å apart in both c ring rotors. This finding suggests that both ATPases have the same gear distance despite their different stoichiometries. The putative proton-binding site at the conserved carboxylate Glu⁶¹ in the chloroplast ATP synthase differs from the sodium-binding site in Ilyobacter. Residues adjacent to the conserved carboxylate show increased hydrophobicity and reduced hydrogen bonding. The crystal structure reflects the protonated form of the chloroplast c ring rotor. We propose that upon deprotonation, the conformation of Glu⁶¹ is changed to another rotamer and becomes fully exposed to the periphery of the ring. Reprotonation of Glu⁶¹ by a conserved arginine in the adjacent a subunit returns the carboxylate to its initial conformation.ATP synthases found in the energy-transducing membranes of bacteria, mitochondria, and chloroplasts catalyze ATP synthesis and ATP hydrolysis coupled with transmembrane proton or sodium ion transport. The enzymes are multi-subunit complexes composed of an extra-membranous catalytic F₁ domain and an interconnected integral membrane F₀ domain. The hydrophilic F₁ domain consists of five different polypeptides with a stoichiometry of α₃β₃γδϵ. Detailed structural information obtained with the mitochondrial enzyme (1–3) in combination with biochemical (4), biophysical (5), and single molecule studies (6–9) revealed that synthesis or hydrolysis of ATP in the F₁ domain is accomplished via a rotary catalytic mechanism. In addition to information on the catalytic mechanism, structure analysis and single molecule studies of the mitochondrial or the chloroplast F₁ complex have also unraveled the molecular mechanism of several F₁-specific inhibitors (10–14). Less detailed information is available on the integral membrane F₀ domain, which consists of three different polypeptides (a, b, and c) and mediates the transfer of protons or sodium ions across the membrane. Subunits a and b were shown to reside at the periphery of a cylindrical complex formed by multiple copies of the c subunit (15–18). The number of c subunits in the cylindrical subcomplex shows substantial variation in different organisms. Ten protomers are found in ATP synthases from yeast, Escherichia coli and Bacillus PS3 (19–21), 11 in Ilyobacter tartaricus, Propionigenium modestum, and Clostridium paradoxum (22–24), 13 in the thermoalkalophilic Bacillus TA2.TA1 (25), 14 in spinach chloroplasts (26), and 15 in the cyanobacterium Spirulina platensis (27). The structure of isolated subunits a, b, and c from E. coli has been studied by mutagenesis analysis and by NMR spectroscopy in a mixed solvent that was suggested to mimic the membrane environment (28–32). These studies showed that subunit a folds with five membrane-spanning helices. The fourth of these helices directly interacts with subunit c and contains a conserved arginine (Arg²¹⁰), which is thought to be involved in proton transfer (33). Subunit b, which is present in two copies in the intact F₀, contains a single transmembrane helix. Cross-linking data support a direct interaction of the two copies of the b subunit (29). Subunit c was studied at two different pH values to obtain the protonated and deprotonated form of a conserved carboxylate (Asp⁶¹ in E. coli) that was shown to be essential for proton transport (34). NMR spectroscopy revealed that the isolated c subunit consists of two long hydrophobic membrane spanning segments connected by a short hydrophilic loop (30, 35). This loop is located close to the γ and ϵ subunit on the F₁ side of the membrane (36, 37). Low resolution x-ray crystallography, cryo-electron microscopy, and atomic force microscopy showed that the membrane-spanning helices of the multiple copies of subunit c in the intact F₀ complex are tightly packed in two concentric rings (19, 22, 26). Atomic resolution of the c ring was recently provided for the Na⁺-translocating F-type ATPase from I. tartaricus (38) and the related Na⁺-translocating V-type ATPase from Enterococcus hirae (39). Rotation of the c ring was demonstrated by cross-linking (18), fluorescence studies (40), and single molecule visualization (41, 42). Based on the structural and biochemical information on F₁ and F₀, different mechanical models have been proposed describing how the rotation of the c ring is coupled to the rotation of the F₁ rotor subunits. This rotation in turn drives sequential conformational shifts at the three catalytic β subunits that result in ATP synthesis (43–45). Vice versa hydrolysis of ATP in the F₁ domain is thought to drive rotation of the γϵc_10–15 subcomplex and transports protons or sodium ions across the membrane.Here we describe the crystal structure of the chloroplast c₁₄ rotor, which is the first structure of an isolated c ring rotor from a proton driven ATPase. The structure was solved by molecular replacement using a tetradecameric search model that was generated from a monomer taken from the I. tartaricus c₁₁ structure. The imposition of noncrystallographic symmetry restraints during refinement substantially improved electron density and structure determination.     

Differential Regulation of Cell Type-specific Apoptosis by Stromelysin-3: A POTENTIAL MECHANISM VIA THE CLEAVAGE OF THE LAMININ RECEPTOR DURING TAIL RESORPTION IN XENOPUS LAEVIS*

Matrix metalloproteinases (MMPs) have been extensively studied because of their functional attributes in development and diseases. However, relatively few in vivo functional studies have been reported on the roles of MMPs in postembryonic organ development. Amphibian metamorphosis is a unique model for studying MMP function during vertebrate development because of its dependence on thyroid hormone (T3) and the ability to easily manipulate this process with exogenous T3. The MMP stromelysin-3 (ST3) is induced by T3, and its expression correlates with cell death during metamorphosis. We have previously shown that ST3 is both necessary and sufficient for larval epithelial cell death in the remodeling intestine. To investigate the roles of ST3 in other organs and especially on different cell types, we have analyzed the effect of transgenic overexpression of ST3 in the tail of premetamorphic tadpoles. We report for the first time that ST3 expression, in the absence of T3, caused significant muscle cell death in the tail of premetamorphic transgenic tadpoles. On the other hand, only relatively low levels of epidermal cell death were induced by precocious ST3 expression in the tail, contrasting what takes place during natural and T3-induced metamorphosis when ST3 expression is high. This cell type-specific apoptotic response to ST3 in the tail suggests distinct mechanisms regulating cell death in different tissues. Furthermore, our analyses of laminin receptor, an in vivo substrate of ST3 in the intestine, suggest that laminin receptor cleavage may be an underlying mechanism for the cell type-specific effects of ST3.The extracellular matrix (ECM),³ the dynamic milieu of the cell microenvironment, plays a critical role in dictating the fate of the cell. The cross-talk between the cell and ECM and the timely catabolism of the ECM are crucial for tissue remodeling during development (1). Matrix metalloproteinases (MMPs), extrinsic proteolytic regulators of the ECM, mediate this process to a large extent. MMPs are a large family of Zn²⁺-dependent endopeptidases potentially capable of cleaving the extracellular as well as nonextracellular proteins (2–9). The MMP superfamily includes collagenases, gelatinases, stromelysins, and membrane-type MMPs based on substrate specificity and domain organization (2–4). MMPs have been implicated to influence a wide range of physiological and pathological processes (10–13). The roles of MMPs appear to be very complex. For example, MMPs have been suggested to play roles in both tumor promotion and suppression (13–19). Unfortunately, relatively few functional studies have been carried out in vivo, especially in relation to the mechanisms involved during vertebrate development.Amphibian metamorphosis presents a fascinating experimental model to study MMP function during postembryonic development. A unique and salient feature of the metamorphic process is the absolute dependence on the signaling of thyroid hormone (20–23). This makes it possible to prevent metamorphosis by simply inhibiting the synthesis of endogenous T3 or to induce precocious metamorphosis by merely adding physiological levels of T3 in the rearing water of premetamorphic tadpoles. Gene expression screens have identified the MMP stromelysin-3 (ST3) as a direct T3 response gene (24–27). Expression studies have revealed a distinct spatial and temporal ST3 expression profile in correlation with metamorphic event, especially cell death (25, 28–31). Organ culture studies on intestinal remodeling have directly substantiated an essential role of ST3 in larval epithelial cell death and ECM remodeling (32). Furthermore, precocious expression of ST3 alone in premetamorphic tadpoles through transgenesis is sufficient to induce ECM remodeling and larval epithelial apoptosis in the tadpole intestine (33). Thus, ST3 appears to be necessary and sufficient for intestinal epithelial cell death during metamorphosis.ST3 was first isolated as a breast cancer-associated gene (34), and unlike most other MMPs, ST3 is secreted as an active protease through a furin-dependent intracellular activation mechanism (35). Like many other MMPs, ST3 is expressed in a number of pathological processes, including most human carcinomas (11, 36–40), as well as in many developmental processes in mammals (10, 34, 41–43), although the physiological and pathological roles of ST3 in vivo are largely unknown in mammals. Interestingly, compared with other MMPs, ST3 has only weak activities toward ECM proteins in vitro but stronger activities against non-ECM proteins like α1 proteinase inhibitor and IGFBP-1 (44–46). Although ST3 may cleave ECM proteins strongly in the in vivo environment, these findings suggest that the cleavage of non-ECM proteins is likely important for its biological roles. Consistently, we have recently identified a cell surface receptor, laminin receptor (LR) as an in vivo substrate of ST3 in the tadpole intestine during metamorphosis (47–49). Analyses of LR expression and cleavage suggest that LR cleavage by ST3 is likely an important mechanism by which ST3 regulates the interaction between the larval epithelial cells and the ECM to induce cell death during intestinal remodeling (47, 48).Here, to investigate the role of ST3 in the apoptosis in other tissues during metamorphosis and whether LR cleavage serves as a mechanism for ST3 to regulate the fate of different cell types, we have analyzed the effects of precocious expression of ST3 in premetamorphic tadpole tail. The tail offers an opportunity to examine the effects of ST3 on different cell types. The epidermis, the fast and slow muscles, and the connective tissue underlying the epidermis in the myotendinous junctions and surrounding the notochord constitute the major tissue types in tail (50). Even though death is the destiny of all these cell types, it is not clear whether they all die through similar or different mechanisms. Microscopic and histochemical analyses have shown that at least the muscle and epidermal cells undergo T3-dependent apoptosis during metamorphosis (23, 29, 51, 52). To study whether ST3 regulates apoptosis of these two cell types, we have made use of the transgenic animals that express a transgenic ST3 under the control of a heat shock-inducible promoter (33). We show that whereas extensive apoptosis is present in both the epidermis and muscles during natural as well as T3-induced metamorphosis, transgenic expression of ST3 induces cell death predominantly in the muscles. Furthermore, we show that LR is expressed in the epidermis and connective tissue but not in muscles of the tadpole tail. More importantly, LR cleavage products are present in the tail during natural metamorphosis but not in transgenic tadpoles overexpressing ST3. These results suggest that ST3 has distinct effects on the epidermis and muscles in the tail, possibly because of the tissue-specific expression and function of LR.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Proteome-wide Substrate Analysis Indicates Substrate Exclusion as a Mechanism to Generate Caspase-7 Versus Caspase-3 Specificity

Caspase-3 and -7 are considered functionally redundant proteases with similar proteolytic specificities. We performed a proteome-wide screen on a mouse macrophage lysate using the N-terminal combined fractional diagonal chromatography technology and identified 46 shared, three caspase-3-specific, and six caspase-7-specific cleavage sites. Further analysis of these cleavage sites and substitution mutation experiments revealed that for certain cleavage sites a lysine at the P5 position contributes to the discrimination between caspase-7 and -3 specificity. One of the caspase-7-specific substrates, the 40 S ribosomal protein S18, was studied in detail. The RPS18-derived P6–P5′ undecapeptide retained complete specificity for caspase-7. The corresponding P6–P1 hexapeptide still displayed caspase-7 preference but lost strict specificity, suggesting that P′ residues are additionally required for caspase-7-specific cleavage. Analysis of truncated peptide mutants revealed that in the case of RPS18 the P4–P1 residues constitute the core cleavage site but that P6, P5, P2′, and P3′ residues critically contribute to caspase-7 specificity. Interestingly, specific cleavage by caspase-7 relies on excluding recognition by caspase-3 and not on increasing binding for caspase-7.Caspases, a family of evolutionarily conserved proteases, mediate apoptosis, inflammation, proliferation, and differentiation by cleaving many cellular substrates (1–3). The apoptotic initiator caspases (caspase-8, -9, and -10) are activated in large signaling platforms and propagate the death signal by cleavage-induced activation of executioner caspase-3 and -7 (4, 5). Most of the cleavage events occurring during apoptosis have been attributed to the proteolytic activity of these two executioner caspases, which can act on several hundreds of proteins (2, 3, 6, 7). The substrate degradomes of the two main executioner caspases have not been determined but their identification is important to gaining greater insight in their cleavage specificity and biological functions.The specificity of caspases was rigorously profiled by using combinatorial tetrapeptide libraries (8), proteome-derived peptide libraries (9), and sets of individual peptide substrates (10, 11). The results of these studies indicate that specificity motifs for caspase-3 and -7 are nearly indistinguishable with the canonical peptide substrate, DEVD, used to monitor the enzymatic activity of both caspase-3 and -7 in biological samples. This overlap in cleavage specificity is manifested in their generation of similar cleavage fragments from a variety of apoptosis-related substrates such as inhibitor of caspase-activated DNase, keratin 18, PARP,1 protein-disulfide isomerase, and Rho kinase I (for reviews, see Refs. 2, 3, and 7). This propagated the view that these two caspases have completely redundant functions during apoptosis. Surprisingly, mice deficient in one of these caspases (as well as mice deficient in both) have distinct phenotypes. Depending on the genetic background of the mice, caspase-3-deficient mice either die before birth (129/SvJ) or develop almost normally (C57BL/6J) (12–14). This suggests that dynamics in the genetic background, such as increased caspase-7 expression, compensate for the functional loss of caspase-3 (15). In the C57BL/6J background, caspase-7 single deficient mice are also viable, whereas caspase-3 and -7 double deficient mice die as embryos, further suggesting redundancy (12–14). However, because caspase-3 and -7 probably arose from gene duplication between the Cephalochordata-Vertebrata diversion (16), they might have acquired different substrate specificities during evolution. Caspase-3 and -7 do exhibit different activities on a few arbitrarily identified natural substrates, including BID, X-linked inhibitor of apoptosis protein, gelsolin, caspase-6, ataxin-7, and co-chaperone p23 (17–20). In addition, caspase-3 generally cleaves more substrates during apoptosis than caspase-7 and therefore appears to be the major executioner caspase. Moreover, a recent report describing caspase-1-dependent activation of caspase-7, but not of caspase-3, in macrophages in response to microbial stimuli supports the idea of a non-redundant function for caspase-7 downstream of caspase-1 (21).Commercially available “caspase-specific” tetrapeptide substrates are widely used for specific caspase detection, but they display substantial promiscuity and cannot be used to monitor individual caspases in cells (22, 23). Detecting proteolysis by measuring the release of C-terminal fluorophores, such as 7-amino-4-methylcoumarin (amc), restricts the specificity of these peptide substrates to non-prime cleavage site residues, which may have hampered the identification of specific cleavage events. To address this limitation, a recently developed proteomics technique, called proteomic identification of protease cleavage sites, was used to map both non-prime and prime preferences for caspase-3 and -7 on a tryptic peptide library (9). However, no clear distinction in peptide recognition motifs between caspase-3 and -7 could be observed (9). Because not all classical caspase cleavage sites are processed (7), structural or post-translational higher order constraints are likely involved in steering the cleavage site selectivity. Peptide-based approaches generally overlook such aspects.We made use of the COFRADIC N-terminal peptide sorting methodology (24–26) to profile proteolytic events of caspase-3 and -7 in a macrophage proteome labeled by triple stable isotope labeling by amino acids in cell culture (SILAC), which allowed direct comparison of peak intensities in peptide MS spectra and consequent quantification of N termini that are equally, preferably, or exclusively generated by the action of caspase-3 or -7 (26, 27). We identified 55 cleavage sites in 48 protein substrates, encompassing mutual, preferred, and unique caspase-3 and -7 cleavage sites.     

Calpain Expression and Activity during Lens Fiber Cell Differentiation

In animal models, the dysregulated activity of calcium-activated proteases, calpains, contributes directly to cataract formation. However, the physiological role of calpains in the healthy lens is not well defined. In this study, we examined the expression pattern of calpains in the mouse lens. Real time PCR and Western blotting data indicated that calpain 1, 2, 3, and 7 were expressed in lens fiber cells. Using controlled lysis, depth-dependent expression profiles for each calpain were obtained. These indicated that, unlike calpain 1, 2, and 7, which were most abundant in cells near the lens surface, calpain 3 expression was strongest in the deep cortical region of the lens. We detected calpain activities in vitro and showed that calpains were active in vivo by microinjecting fluorogenic calpain substrates into cortical fiber cells. To identify endogenous calpain substrates, membrane/cytoskeleton preparations were treated with recombinant calpain, and cleaved products were identified by two-dimensional difference electrophoresis/mass spectrometry. Among the calpain substrates identified by this approach was αII-spectrin. An antibody that specifically recognized calpain-cleaved spectrin was used to demonstrate that spectrin is cleaved in vivo, late in fiber cell differentiation, at or about the time that lens organelles are degraded. The generation of the calpain-specific spectrin cleavage product was not observed in lens tissue from calpain 3-null mice, indicating that calpain 3 is uniquely activated during lens fiber differentiation. Our data suggest a role for calpains in the remodeling of the membrane cytoskeleton that occurs with fiber cell maturation.Calpains comprise a family of cysteine proteases named for the calcium dependence of the founder members of the family, the ubiquitously expressed enzymes, calpain 1 (μ-calpain) and calpain 2 (m-calpain). The calpain family includes more than a dozen members with sequence relatedness to the catalytic subunits of calpain 1 and 2. Calpains have a modular domain architecture. By convention, the family is subdivided into classical and nonclassical calpains, according to the presence or absence, respectively, of a calcium-binding penta-EF-hand module in domain IV of the protein (1). Classical calpains include calpain 1, 2, 3, 8, 9, and 11. Nonclassical calpains include calpain 5, 6, 7, 10, 12, 13, and 14.Transgenic and gene knock-out approaches in mice have demonstrated an essential role for calpains during embryonic development. Knock-out of the small regulatory subunit (Capn4) results in embryonic lethality (2, 3). Similarly, inactivation of the Capn2 gene blocks development between the morula and blastocyst stage (4). In humans, mutations in CAPN3 underlie limb-girdle muscular dystrophy-2A, and polymorphisms in CAPN10 may predispose to type 2 diabetes mellitus (5, 6).Even under conditions of calcium overload, where calpains are presumably activated maximally, only a subset (<5%) of cellular proteins are hydrolyzed (7). Calpains typically cleave their substrates at a limited number of sites to generate large polypeptide fragments that, in many cases, retain bioactivity. Thus, under physiological conditions, calpains probably participate in the regulation of protein function rather than in non-specific protein degradation.More than 100 proteins have been shown to serve as calpain substrates in vitro, including cytoskeletal proteins (8), signal transduction molecules (9), ion channels (10), and receptors (11). In vivo, calpains are believed to function in myoblast fusion (12), long term potentiation (13), and cellular mobility (14). Unregulated calpain activity, secondary to intracellular calcium overload, is associated with several pathological conditions, including Alzheimer disease (15), animal models of cataract (16), myocardial (17), and cerebral ischemia (18).In addition to their domain structure, calpains are often classified according to their tissue expression patterns. Calpain 1, 2, and 10 are widely expressed in mammalian tissues, but other members of the calpain family show tissue-specific expression patterns. Calpain 8, for example, is a stomach-specific calpain (19), whereas expression of calpain 9 is restricted to tissues of the digestive tract (20). The expression of calpain 3 was originally thought to be limited to skeletal muscle (21), but splice variants of calpain 3 have since been detected in a range of tissues. At least 12 isoforms of calpain 3 have been described in rodents (22), of which several are expressed in the mammalian eye, including Lp82 (lens), Cn94 (cornea), and Rt88 (retina) (23).Calpains have been studied intensively in the ocular lens because of their suspected involvement in lens opacification (cataract). Calpain-mediated proteolysis of lens crystallin proteins causes increased light scatter (24). Unregulated activation of calpains is observed in rodent cataract models (25), where calpain-mediated degradation of crystallin proteins (26) and cytoskeletal elements (27) is commonly observed. Calpain inhibitors are effective in delaying or preventing cataract in vitro (28, 29) and in vivo (30, 31).It is likely, however, that calpains have important physiological roles in the lens beyond their involvement in tissue pathology. Terminal differentiation of lens fiber cells involves a series of profound morphological and biochemical transformations. For example, differentiating lens fiber cells undergo an enormous (>100-fold) increase in cell length, accompanied by extensive remodeling of the plasma membrane system (32). Early in the differentiation process, fusion pores are established between cells, as neighboring fibers are incorporated into the lens syncytium (33). A later stage of fiber cell differentiation involves the dissolution of all intracellular organelles, a process that is thought to eliminate light-scattering particles from the light path and contribute to the transparency of the tissue (34). Any or all of these phenomena might require the developmentally regulated activation of calpains. This is consistent with our previous observation that in calpain 3 knock-out mice the transition zone is altered, suggesting a change in the differentiation program (35).In the current study, therefore, we examined the depth-dependent expression pattern and activity of calpains in the mouse lens. Fluorogenic substrates were microinjected into the intact lens to visualize calpain activity directly, and proteomic approaches were used to identify endogenous calpain substrates. The cleavage pattern of one of these, αII-spectrin, was examined in detail. Immunocytochemical and immunoblot analysis with wild type and calpain 3-null lenses indicated that αII-spectrin is a specific calpain 3 substrate in maturing lens fiber cells. Together, the data suggest that calpains are activated relatively late in fiber cell differentiation and may contribute to the remodeling of the membrane cytoskeleton that accompanies fiber cell maturation.