Advances in apoptosis research

Apoptosis, also called programmed cell death, has attracted great attention in recent years. After its discovery by Carl Vogt in 1842, apoptosis research was dormant for more than a century. Its rediscovery in the second half of this century, and the coining of the term apoptosis in 1972 by Kerr, Wyllie, and Currie, ignited an unparalleled interest in this field of science. The number of publications related to apoptosis has been growing exponentially every year ever since. This is mainly due to three major advances, two of which have been made recently and one that is currently seen. First, studies with the small nematode Caenorhabditis elegans have identified a number of apoptosis regulating genes—the first evidence that cell death is an active process under genetic control. Many of these genes have mammalian homologs that, like their worm counterparts, seem to regulate mammalian apoptosis. Second, elucidation of the signal transduction pathways of apoptosis has lead especially to the identification of specific death signaling molecules such as a new family of cysteine proteases, the caspases. Third, it has now become clear that many diseases are characterized by dysregulation of apoptotic programs. Many of these programs involve a family of receptors and their ligands, the death receptor/ligand family. The hope now is to interfere with apoptosis regulation in these systems and to develop new therapeutic concepts.

Caspase activation: The induced-proximity model

Members of the caspase family of proteases transmit the events that lead to apoptosis of animal cells. Distinct members of the family are involved in both the initiation and execution phases of cell death, with the initiator caspases being recruited to multicomponent signaling complexes. Initiation of apoptotic events depends on the ability of the signaling complexes to generate an active protease. The mechanism of activation of the caspases that constitute the different apoptosis-signaling complexes can be explained by an unusual property of the caspase zymogens to autoprocess to an active form. This autoprocessing depends on intrinsic activity that resides in the zymogens of the initiator caspases. We review evidence for a hypothesis—the induced-proximity model—that describes how the first proteolytic signal is produced after adapter-mediated clustering of initiator caspase zymogens.

Structural aspects of activation pathways of aspartic protease zymogens and viral 3C protease precursors

The three-dimensional structures of the inactive protein precursors (zymogens) of the serine, cysteine, aspartic, and metalloprotease classes of proteolytic enzymes are known. Comparisons of these structures with those of the mature, active proteases reveal that, in general, the preformed, active conformations of the residues involved in catalysis are rendered sterically inaccessible to substrates by the residues of the zymogens’ N-terminal extensions or prosegments. The prosegments interact in nonsubstrate-like fashions with the residues of the active sites in most of the cases. The gastric aspartic proteases have a well-characterized zymogen conversion pathway. Structures of human progastricsin, the inactive intermediate 2, and active human pepsin are known and have been used to define the conversion pathway. The structure of the zymogen precursor of plasmepsin II, the malarial aspartic protease, shows a new twist on the mode of inactivation used by the gastric zymogens. The prosegment of proplasmepsin disrupts the active conformation of the two catalytic aspartic acid residues by inducing a major reorientation of the two domains of the mature protease. The picornaviral 2A and 3C proteases have a chymotrypsin-like tertiary structure but with a cysteine nucleophile. These enzymes cleave themselves from the viral polyprotein in cis (intramolecular cleavage) and carry out trans cleavages of other scissile peptides important for the virus life cycle. Although the structure of the precursor viral polyprotein is unknown, it probably resembles the organization of the proenzymes of the bacterial serine proteases, subtilisin, and α-lytic protease. Cleavage of the prosegment is known to occur in cis for these precursor molecules.

Kinetic stability as a mechanism for protease longevity

The folding of the extracellular serine protease, α-lytic protease (αLP; EC 3.4.21.12) reveals a novel mechanism for stability that appears to lead to a longer functional lifetime for the protease. For αLP, stability is based not on thermodynamics, but on kinetics. Whereas this has required the coevolution of a pro region to facilitate folding, the result has been the optimization of native-state properties independent of their consequences on thermodynamic stability. Structural and mutational data lead to a model for catalysis of folding in which the pro region binds to a conserved β-hairpin in the αLP C-terminal domain, stabilizing the folding transition state and the native state. The pro region is then proteolytically degraded, leaving the active αLP trapped in a metastable conformation. This metastability appears to be a consequence of pressure to evolve properties of the native state, including a large, highly cooperative barrier to unfolding, and extreme rigidity, that reduce susceptibility to proteolytic degradation. In a test of survival under highly proteolytic conditions, homologous mammalian proteases that have not evolved kinetic stability are much more rapidly degraded than αLP. Kinetic stability as a means to longevity is likely to be a mechanism conserved among the majority of extracellular bacterial pro-proteases and may emerge as a general strategy for intracellular eukaryotic proteases subject to harsh conditions as well.

Cysteine protease inhibitors as chemotherapy: Lessons from a parasite target

Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite’s lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

How the protease thrombin talks to cells

How does a protease act like a hormone to regulate cellular functions? The coagulation protease thrombin (EC 3.4.21.5) activates platelets and regulates the behavior of other cells by means of G protein-coupled protease-activated receptors (PARs). PAR1 is activated when thrombin binds to and cleaves its amino-terminal exodomain to unmask a new receptor amino terminus. This new amino terminus then serves as a tethered peptide ligand, binding intramolecularly to the body of the receptor to effect transmembrane signaling. The irreversibility of PAR1’s proteolytic activation mechanism stands in contrast to the reversible ligand binding that activates classical G protein-coupled receptors and compels special mechanisms for desensitization and resensitization. In endothelial cells and fibroblasts, activated PAR1 rapidly internalizes and then sorts to lysosomes rather than recycling to the plasma membrane as do classical G protein-coupled receptors. This trafficking behavior is critical for termination of thrombin signaling. An intracellular pool of thrombin receptors refreshes the cell surface with naïve receptors, thereby maintaining thrombin responsiveness. Thus cells have evolved a trafficking solution to the signaling problem presented by PARs. Four PARs have now been identified. PAR1, PAR3, and PAR4 can all be activated by thrombin. PAR2 is activated by trypsin and by trypsin-like proteases but not by thrombin. Recent studies with knockout mice, receptor-activating peptides, and blocking antibodies are beginning to define the role of these receptors in vivo.

Chaperone rings in protein folding and degradation

Chaperone rings play a vital role in the opposing ATP-mediated processes of folding and degradation of many cellular proteins, but the mechanisms by which they assist these life and death actions are only beginning to be understood. Ring structures present an advantage to both processes, providing for compartmentalization of the substrate protein inside a central cavity in which multivalent, potentially cooperative interactions can take place between the substrate and a high local concentration of binding sites, while access of other proteins to the cavity is restricted sterically. Such restriction prevents outside interference that could lead to nonproductive fates of the substrate protein while it is present in non-native form, such as aggregation. At the step of recognition, chaperone rings recognize different motifs in their substrates, exposed hydrophobicity in the case of protein-folding chaperonins, and specific “tag” sequences in at least some cases of the proteolytic chaperones. For both folding and proteolytic complexes, ATP directs conformational changes in the chaperone rings that govern release of the bound polypeptide. In the case of chaperonins, ATP enables a released protein to pursue the native state in a sequestered hydrophilic folding chamber, and, in the case of the proteases, the released polypeptide is translocated into a degradation chamber. These divergent fates are at least partly governed by very different cooperating components that associate with the chaperone rings: that is, cochaperonin rings on one hand and proteolytic ring assemblies on the other. Here we review the structures and mechanisms of the two types of chaperone ring system.

Reverse biochemistry: Use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue

Serine proteases of the chymotrypsin fold are of great interest because they provide detailed understanding of their enzymatic properties and their proposed role in a number of physiological and pathological processes. We have been developing the macromolecular inhibitor ecotin to be a “fold-specific” inhibitor that is selective for members of the chymotrypsin-fold class of proteases. Inhibition of protease activity through the use of wild-type and engineered ecotins results in inhibition of rat prostate differentiation and retardation of the growth of human PC-3 prostatic cancer tumors. In an effort to identify the proteases that may be involved in these processes, reverse transcription–PCR with PC-3 poly(A)+ mRNA was performed by using degenerate oligonucleotide primers. These primers were designed by using conserved protein sequences unique to chymotrypsin-fold serine proteases. Five proteases were identified: urokinase-type plasminogen activator, factor XII, protein C, trypsinogen IV, and a protease that we refer to as membrane-type serine protease 1 (MT-SP1). The cloning and characterization of the MT-SP1 cDNA shows that it encodes a mosaic protein that contains a transmembrane signal anchor, two CUB domains, four LDLR repeats, and a serine protease domain. Northern blotting shows broad expression of MT-SP1 in a variety of epithelial tissues with high levels of expression in the human gastrointestinal tract and the prostate. A His-tagged fusion of the MT-SP1 protease domain was expressed in Escherichia coli, purified, and autoactivated. Ecotin and variant ecotins are subnanomolar inhibitors of the MT-SP1 activated protease domain, suggesting a possible role for MT-SP1 in prostate differentiation and the growth of prostatic carcinomas.

Induction of Acclimative Proteolysis of the Light-Harvesting Chlorophyll a/b Protein of Photosystem II in Response to Elevated Light Intensities1

Most plants have the ability to respond to fluctuations in light to minimize damage to the photosynthetic apparatus. A proteolytic activity has been discovered that is involved in the degradation of the major light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCII) when the antenna size of photosystem II is reduced upon acclimation of plants from low to high light intensities. This ATP-dependent proteolytic activity is of the serine or cysteine type and is associated with the outer membrane surface of the stroma-exposed thylakoid regions. The identity of the protease is not known, but it does not correspond to the recently identified chloroplast ATP-dependent proteases Clp and FtsH, which are homologs to bacterial enzymes. The acclimative response shows a delay of 2 d after transfer of the leaves to high light. This lag period was shown to be attributed to expression or activation of the responsible protease. Furthermore, the LHCII degradation was found to be regulated at the substrate level. The degradation process involves lateral migration of LHCII from the appressed to the nonappressed thylakoid regions, which is the location for the responsible protease. Phosphorylated LHCII was found to be a poor substrate for degradation in comparison with the unphosphorylated form of the protein. The relationship between LHCII degradation and other regulatory proteolytic processes in the thylakoid membrane, such as D1-protein degradation, is discussed.

Proteasome Inhibitors Prevent Tracheary Element Differentiation in Zinnia Mesophyll Cell Cultures1

To determine whether proteasome activity is required for tracheary element (TE) differentiation, the proteasome inhibitors clasto-lactacystin β-lactone and carbobenzoxy-leucinyl-leucinyl-leucinal (LLL) were used in a zinnia (Zinnia elegans) mesophyll cell culture system. The addition of proteasome inhibitors at the time of culture initiation prevented differentiation otherwise detectable at 96 h. Inhibition of the proteasome at 48 h, after cellular commitment to differentiation, did not alter the final percentage of TEs compared with controls. However, proteasome inhibition at 48 h delayed the differentiation process by approximately 24 h, as indicated by examination of both morphological markers and the expression of putative autolytic proteases. These results indicate that proteasome function is required both for induction of TE differentiation and for progression of the TE program in committed cells. Treatment at 48 h with LLL but not clasto-lactacystin β-lactone resulted in partial uncoupling of autolysis from differentiation. Results from gel analysis of protease activity suggested that the observed incomplete autolysis was due to the ability of LLL to inhibit TE cysteine proteases.

Mechanistic coupling of protease signaling and initiation of coagulation by tissue factor

The crucial role of cell signaling in hemostasis is clearly established by the action of the downstream coagulation protease thrombin that cleaves platelet-expressed G-protein-coupled protease activated receptors (PARs). Certain PARs are cleaved by the upstream coagulation proteases factor Xa (Xa) and the tissue factor (TF)–factor VIIa (VIIa) complex, but these enzymes are required at high nonphysiological concentrations and show limited recognition specificity for the scissile bond of target PARs. However, defining a physiological mechanism of PAR activation by upstream proteases is highly relevant because of the potent anti-inflammatory in vivo effects of inhibitors of the TF initiation complex. Activation of substrate factor X (X) by the TF–VIIa complex is here shown to produce enhanced cell signaling in comparison to the TF–VIIa complex alone, free Xa, or Xa that is generated in situ by the intrinsic activation complex. Macromolecular assembly of X into a ternary complex of TF–VIIa–X is required for proteolytic conversion to Xa, and product Xa remains transiently associated in a TF–VIIa–Xa complex. By trapping this complex with a unique inhibitor that preserves Xa activity, we directly show that Xa in this ternary complex efficiently activates PAR-1 and -2. These experiments support the concept that proinflammatory upstream coagulation protease signaling is mechanistically coupled and thus an integrated part of the TF–VIIa-initiated coagulation pathway, rather than a late event during excessive activation of coagulation and systemic generation of proteolytic activity.

The complete genome of the crenarchaeon Sulfolobus solfataricus P2

The genome of the crenarchaeon Sulfolobus solfataricus P2 contains 2,992,245 bp on a single chromosome and encodes 2,977 proteins and many RNAs. One-third of the encoded proteins have no detectable homologs in other sequenced genomes. Moreover, 40% appear to be archaeal-specific, and only 12% and 2.3% are shared exclusively with bacteria and eukarya, respectively. The genome shows a high level of plasticity with 200 diverse insertion sequence elements, many putative nonautonomous mobile elements, and evidence of integrase-mediated insertion events. There are also long clusters of regularly spaced tandem repeats. Different transfer systems are used for the uptake of inorganic and organic solutes, and a wealth of intracellular and extracellular proteases, sugar, and sulfur metabolizing enzymes are encoded, as well as enzymes of the central metabolic pathways and motility proteins. The major metabolic electron carrier is not NADH as in bacteria and eukarya but probably ferredoxin. The essential components required for DNA replication, DNA repair and recombination, the cell cycle, transcriptional initiation and translation, but not DNA folding, show a strong eukaryal character with many archaeal-specific features. The results illustrate major differences between crenarchaea and euryarchaea, especially for their DNA replication mechanism and cell cycle processes and their translational apparatus.

Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death

The inhibitor of apoptosis (IAP) family of anti-apoptotic proteins regulate programmed cell death and/or apoptosis. One such protein, X-linked IAP (XIAP), inhibits the activity of the cell death proteases, caspase-3, -7, and -9. In this study, using constitutively active mutants of caspase-3, we found that XIAP promotes the degradation of active-form caspase-3, but not procaspase-3, in living cells. The XIAP mutants, which cannot interact with caspase-3, had little or no activity of promoting the degradation of caspase-3. RING finger mutants of XIAP also could not promote the degradation of caspase-3. A proteasome inhibitor suppressed the degradation of caspase-3 by XIAP, suggesting the involvement of a ubiquitin-proteasome pathway in the degradation. An in vitro ubiquitination assay revealed that XIAP acts as a ubiquitin-protein ligase for caspase-3. Caspase-3 was ubiquitinated in the presence of XIAP in living cells. Both the association of XIAP with caspase-3 and the RING finger domain of XIAP were essential for ubiquitination. Finally, the RING finger mutants of XIAP were less effective than wild-type XIAP at preventing apoptosis induced by overexpression of either active-form caspase-3 or Fas. These results demonstrate that the ubiquitin-protein ligase activity of XIAP promotes the degradation of caspase-3, which enhances its anti-apoptotic effect.

Differential suppression by protease inhibitors and cytokines of apoptosis induced by wild-type p53 and cytotoxic agents.

Apoptosis induced in myeloid leukemic cells by wild-type p53 was suppressed by different cleavage-site directed protease inhibitors, which inhibit interleukin-1 beta-converting enzyme-like, granzyme B and cathepsins B and L proteases. Apoptosis was also suppressed by the serine and cysteine protease inhibitor N-tosyl-L-phenylalanine chloromethylketone (TPCK) [corrected], but not by other serine or cysteine protease inhibitors including N alpha-p-tosyl-L-lysine chloromethylketone (TLCK), E64, pepstatin A, or chymostatin. Protease inhibitors suppressed induction of apoptosis by gamma-irradiation and cycloheximide but not by doxorubicin, vincristine, or withdrawal of interleukin 3 from interleukin 3-dependent 32D non-malignant myeloid cells. Induction of apoptosis in normal thymocytes by gamma-irradiation or dexamethasone was also suppressed by the cleavage-site directed protease inhibitors, but in contrast to the myeloid leukemic cells apoptosis in thymocytes was suppressed by TLCK but not by TPCK. The results indicate that (i) inhibitors of interleukin-1 beta-converting enzyme-like proteases and some other protease inhibitors suppressed induction of apoptosis by wild-type p53 and certain p53-independent pathways of apoptosis; (ii) the protease inhibitors together with the cytokines interleukin 6 and interferon-gamma or the antioxidant butylated hydroxyanisole gave a cooperative protection against apoptosis; (iii) these protease inhibitors did not suppress induction of apoptosis by some cytotoxic agents or by viability-factor withdrawal from 32D cells, whereas these pathways of apoptosis were suppressed by cytokines; (iv) there are cell type differences in the proteases involved in apoptosis; and (v) there are multiple pathways leading to apoptosis that can be selectively induced and suppressed by different agents.

p53-dependent cell cycle arrest induced by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal in platelet-derived growth factor-stimulated human fibroblasts.

Proteases are known to play important roles in cell growth control, although the underlying mechanisms are still poorly understood. Here we show that the protease inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal induced cell cycle arrest in platelet-derived growth factor-stimulated human fibroblasts at the G1/S boundary of the cell cycle by inhibiting the proteasome. Inhibition of the proteasome resulted in accumulation of the tumor suppressor p53, which was followed by an increase in the amount of the cyclin-dependent kinase-inhibitor p21. As a consequence, both phosphorylation and activity of the cyclin-dependent kinase 2/cyclin E complex were inhibited. We further observed that the retinoblastoma gene product, pRb, remained in the hypophosphorylated state, thus preventing cells from progression into the S-phase. These studies strongly support the hypothesis that the proteasome is a key regulator in the G1-phase of cell cycle progression.