Activation of distinct caspase-like proteases by Fas and reaper in Drosophila cells

The cytoplasmic region of Fas, a mammalian death factor receptor, shares a limited homology with reaper, an apoptosis-inducing protein in Drosophila. Expression of either the Fas cytoplasmic region (FasC) or of reaper in Drosophila cells caused cell death. The death process induced by FasC or reaper was inhibited by crmA or p35, suggesting that its death process is mediated by caspase-like proteases. Both Ac-YVAD aldehyde and Ac-DEVD aldehyde, specific inhibitors of caspase 1- and caspase 3-like proteases, respectively, inhibited the FasC-induced death of Drosophila cells. However, the cell death induced by reaper was inhibited by Ac-DEVD aldehyde, but not by Ac-YVAD aldehyde. A caspase 1-like protease activity that preferentially recognizes the YVAD sequence gradually increased in the cytosolic fraction of the FasC-activated cells, whereas the caspase 3-like protease activity recognizing the DEVD sequence was observed in the reaper-activated cells. Partial purification and biochemical characterization of the proteases indicated that there are at least three distinct caspase-like proteases in Drosophila cells, which are differentially activated by FasC and reaper. The conservation of the Fas-death signaling pathway in Drosophila cells, which is distinct from that for reaper, may indicate that cell death in Drosophila is controlled not only by the reaper suicide gene, but also by a Fas-like killer gene.

Molecular ordering of the Fas-apoptotic pathway: The Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases

The Fas/APO-1-receptor associated cysteine protease Mch5 (MACH/FLICE) is believed to be the enzyme responsible for activating a protease cascade after Fas-receptor ligation, leading to cell death. The Fas-apoptotic pathway is potently inhibited by the cowpox serpin CrmA, suggesting that Mch5 could be the target of this serpin. Bacterial expression of proMch5 generated a mature enzyme composed of two subunits, which are derived from the precursor proenzyme by processing at Asp-227, Asp-233, Asp-391, and Asp-401. We demonstrate that recombinant Mch5 is able to process/activate all known ICE/Ced-3-like cysteine proteases and is potently inhibited by CrmA. This contrasts with the observation that Mch4, the second FADD-related cysteine protease that is also able to process/activate all known ICE/Ced-3-like cysteine proteases, is poorly inhibited by CrmA. These data suggest that Mch5 is the most upstream protease that receives the activation signal from the Fas-receptor to initiate the apoptotic protease cascade that leads to activation of ICE-like proteases (TX, ICE, and ICE-relIII), Ced-3-like proteases (CPP32, Mch2, Mch3, Mch4, and Mch6), and the ICH-1 protease. On the other hand, Mch4 could be a second upstream protease that is responsible for activation of the same protease cascade in CrmA-insensitive apoptotic pathways.

BAX-induced cell death may not require interleukin 1β-converting enzyme-like proteases

Expression of BAX, without another death stimulus, proved sufficient to induce a common pathway of apoptosis. This included the activation of interleukin 1β-converting enzyme (ICE)-like proteases with cleavage of the endogenous substrates poly(ADP ribose) polymerase and D4-GDI (GDP dissociation inhibitor for the rho family), as well as the fluorogenic peptide acetyl-Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC). The inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) successfully blocked this protease activity and prevented FAS-induced death but not BAX-induced death. Blocking ICE-like protease activity prevented the cleavage of nuclear and cytosolic substrates and the DNA degradation that followed BAX induction. However, the fall in mitochondrial membrane potential, production of reactive oxygen species, cytoplasmic vacuolation, and plasma membrane permeability that are downstream of BAX still occurred. Thus, BAX-induced alterations in mitochondrial function and subsequent cell death do not apparently require the known ICE-like proteases.

Unexpected crucial role of residue 225 in serine proteases

Residue 225 in serine proteases of the chymotrypsin family is Pro or Tyr in more than 95% of nearly 300 available sequences. Proteases with Y225 (like some blood coagulation and complement factors) are almost exclusively found in vertebrates, whereas proteases with P225 (like degradative enzymes) are present from bacteria to human. Saturation mutagenesis of Y225 in thrombin shows that residue 225 affects ligand recognition up to 60,000-fold. With the exception of Tyr and Phe, all residues are associated with comparable or greatly reduced catalytic activity relative to Pro. The crystal structures of three mutants that differ widely in catalytic activity (Y225F, Y225P, and Y225I) show that although residue 225 makes no contact with substrate, it drastically influences the shape of the water channel around the primary specificity site. The activity profiles obtained for thrombin also suggest that the conversion of Pro to Tyr or Phe documented in the vertebrates occurred through Ser and was driven by a significant gain (up to 50-fold) in catalytic activity. In fact, Ser and Phe are documented in 4% of serine proteases, which together with Pro and Tyr account for almost the entire distribution of residues at position 225. The unexpected crucial role of residue 225 in serine proteases explains the evolutionary selection of residues at this position and shows that the structural determinants of protease activity and specificity are more complex than currently believed. These findings have broad implications in the rational design of enzymes with enhanced catalytic properties.

Alkaline-mediated differential interaction (AMDI): A simple automatable single-nucleotide polymorphism assay

The key requirements for high-throughput single-nucleotide polymorphism (SNP) typing of DNA samples in large-scale disease case-control studies are automatability, simplicity, and robustness, coupled with minimal cost. In this paper we describe a fluorescence technique for the detection of SNPs that have been amplified by using the amplification refractory mutation system (ARMS)-PCR procedure. Its performance was evaluated using 32 sequence-specific primer mixes to assign the HLA-DRB alleles to 80 lymphoblastoid cell line DNAs chosen from our database for their diversity. All had been typed previously by alternative methods, either direct sequencing or gel electrophoresis. We believe the detection system that we call AMDI (alkaline-mediated differential interaction) satisfies the above criteria and is suitable for general high-throughput SNP typing.

Vps41p Function in the Alkaline Phosphatase Pathway Requires Homo-oligomerization and Interaction with AP-3 through Two Distinct Domains

Transport of proteins through the ALP (alkaline phosphatase) pathway to the vacuole requires the function of the AP-3 adaptor complex and Vps41p. However, unlike other adaptor protein–dependent pathways, the ALP pathway has not been shown to require additional accessory proteins or coat proteins, such as membrane recruitment factors or clathrin. Two independent genetic approaches have been used to identify new mutants that affect transport through the ALP pathway. These screens yielded new mutants in both VPS41 and the four AP-3 subunit genes. Two new VPS41 alleles exhibited phenotypes distinct from null mutants of VPS41, which are defective in vacuolar morphology and protein transport through both the ALP and CPY sorting pathways. The new alleles displayed severe ALP sorting defects, normal vacuolar morphology, and defects in ALP vesicle formation at the Golgi complex. Sequencing analysis of these VPS41 alleles revealed mutations encoding amino acid changes in two distinct domains of Vps41p: a conserved N-terminal domain and a C-terminal clathrin heavy-chain repeat (CHCR) domain. We demonstrate that the N-terminus of Vps41p is required for binding to AP-3, whereas the C-terminal CHCR domain directs homo-oligomerization of Vps41p. These data indicate that a homo-oligomeric form of Vps41p is required for the formation of ALP containing vesicles at the Golgi complex via interactions with AP-3.

A Novel Alkaline α-Galactosidase from Melon Fruit with a Substrate Preference for Raffinose1

The cucurbits translocate the galactosyl-sucrose oligosaccharides raffinose and stachyose, therefore, α-galactosidase (α-d-galactoside galactohydrolase, EC 3.2.1.22) is expected to function as the initial enzyme of photoassimilate catabolism. However, the previously described alkaline α-galactosidase is specific for the tetrasaccharide stachyose, leaving raffinose catabolism in these tissues as an enigma. In this paper we report the partial purification and characterization of three α-galactosidases, including a novel alkaline α-galactosidase (form I) from melon (Cucumis melo) fruit tissue. The form I enzyme showed preferred activity with raffinose and significant activity with stachyose. Other unique characteristics of this enzyme, such as weak product inhibition by galactose (in contrast to the other α-galactosidases, which show stronger product inhibition), also impart physiological significance. Using raffinose and stachyose as substrates in the assays, the activities of the three α-galactosidases (alkaline form I, alkaline form II, and the acid form) were measured at different stages of fruit development. The form I enzyme activity increased during the early stages of ovary development and fruit set, in contrast to the other α-galactosidase enzymes, both of which declined in activity during this period. In the mature, sucrose-accumulating mesocarp, the alkaline form I enzyme was the major α-galactosidase present. We also observed hydrolysis of raffinose at alkaline conditions in enzyme extracts from other cucurbit sink tissues, as well as from young Coleus blumei leaves. Our results suggest different physiological roles for the α-galactosidase forms in the developing cucurbit fruit, and show that the newly discovered enzyme plays a physiologically significant role in photoassimilate partitioning in cucurbit sink tissue.

Catalytic efficiency and vitality of HIV-1 proteases from African viral subtypes

The vast majority of HIV-1 infections in Africa are caused by the A and C viral subtypes rather than the B subtype prevalent in the United States and Western Europe. Genomic differences between subtypes give rise to sequence variations in the encoded proteins, including the HIV-1 protease. Because some amino acid polymorphisms occur at sites that have been associated with drug resistance in the B subtype, it is important to assess the effectiveness of protease inhibitors that have been developed against different subtypes. Here we report the enzymatic characterization of HIV-1 proteases with sequences found in drug-naïve Ugandan adults. The A protease used in these studies differs in seven positions (I13V/E35D/M36I/R41K/R57K/H69K/L89M) in relation to the consensus B subtype protease. Another protease containing a subset of these amino acid polymorphisms (M36I/R41K/H69K/L89M), which are found in subtype C and other HIV subtypes, also was studied. Both proteases were found to have similar catalytic constants, kcat, as the B subtype. The C subtype protease displayed lower Km values against two different substrates resulting in a higher (2.4-fold) catalytic efficiency than the B subtype protease. Indinavir, ritonavir, saquinavir, and nelfinavir inhibit the A and C subtype proteases with 2.5–7-fold and 2–4.5-fold weaker Kis than the B subtype. When all factors are taken into consideration it is found that the C subtype protease has the highest vitality (4–11 higher than the B subtype) whereas the A subtype protease exhibits values ranging between 1.5 and 5. These results point to a higher biochemical fitness of the A and C proteases in the presence of existing inhibitors.

Residue 225 determines the Na(+)-induced allosteric regulation of catalytic activity in serine proteases.

Residue 225 in serine proteases is typically Pro or Tyr and specifies an important and unanticipated functional aspect of this class of enzymes. Proteases with Y225, like thrombin, are involved in highly specialized functions like blood coagulation and complement that are exclusively found in vertebrates. In these proteases, the catalytic activity is enhanced allosterically by Na+ binding. Proteases with P225, like trypsin, are typically involved in digestive functions and are also found in organisms as primitive as eubacteria. These proteases have no requirement for Na+ or other monovalent cations. The molecular origin of this physiologically important difference is remarkably simple and is revealed by a comparison of the Na+ binding loop of thrombin with the homologous region of trypsin. The carbonyl O atom of residue 224 makes a key contribution to the coordination shell of the bound Na+ in thrombin, but is oriented in a manner incompatible with Na+ binding in trypsin because of constraints imposed by P225 on the protein backbone. Pro at position 225 is therefore incompatible with Na+ binding and is a direct predictor of the lack of allosteric regulation in serine proteases. To directly test this hypothesis, we have engineered the thrombin mutant Y225P. This mutant has lost the ability to bind Na+ and behaves like the allosteric slow (Na(+)-free) form. The Na(+)-induced allosteric regulation also bears on the molecular evolution of serine proteases. A strong correlation exists between residue 225 and the codon used for the active site S195. Proteases with P225 typically use a TCN codon for S195, whereas proteases with Y225 use an AGY codon. It is proposed that serine proteases evolved from two main lineages: (i) TCN/P225 with a trypsin-like ancestor and (ii) AGY/Y225 with a thrombin-like ancestor. We predict that the Na(+)-induced allosteric regulation of catalytic activity can be introduced in the TCN/P225 lineage using the P225Y replacement.

Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis.

Although proteases related to the interleukin 1 beta-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cell-free model system for apoptosis (S/M extracts). First, we compared the substrate specificities of two recombinant human IRPs, CPP32 and Mch2 alpha. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Mch2 alpha also cleaved recombinant and nuclear lamin A at a conserved VEID decreases NG sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, CPP32 did not cleave lamin A. The cleavage of lamin A by Mch2 alpha and by S/M extracts was inhibited by millimolar concentrations of Zn2+, which had a minimal effect on cleavage of poly (ADP-ribose) polymerase by CPP32 and by S/M extracts. We also found that N-(acetyltyrosinylvalinyl-N epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone, which derivatizes the larger subunit of active ICE, can affinity label up to five active IRPs in S/M extracts. Together, these observations indicate that the processing of nuclear proteins in apoptosis involves multiple IRPs having distinct preferences for their apoptosis-associated substrates.

The precursor of PsaD assembles into the photosystem I complex in two steps.

The present study addresses the assembly in the chloroplast thylakoid membranes of PsaD, a peripheral membrane protein of the photosystem I complex. Located on the stromal side of the thylakoids, PsaD was found to assemble in vitro into the membranes in its precursor (pre-PsaD) and also in its mature (PsaD) form. Newly assembled unprocessed pre-PsaD was resistant to NaBr and alkaline wash. Yet it was sensitive to proteolytic digestion. In contradistinction, when the assembled precursor was processed, the resulting mature PsaD was resistant to proteases to the same extent as endogenous [correction of endogeneous] PsaD. The accumulation of protease-resistant PsaD in the thylakoids correlated with the increase of mature-PsaD in the membranes. This protection of mature PsaD from proteolysis could not be observed when PsaD was in a soluble form-i.e. not assembled within the thylakoids. The data suggest that pre-PsaD assembles to the membranes and only in a second step processing takes place. The observation that the assembly of pre-PsaD is affected by salts to a much lesser extent than that of mature-PsaD supports a two-step assembly of pre-PsaD.

The contrasting roles of ICE family proteases and interleukin-1beta in apoptosis induced by trophic factor withdrawal and by copper/zinc superoxide dismutase down-regulation.

We compare here the mechanisms of apoptotic death of PC12 cells induced by down-regulation of Cu2+,Zn2+ superoxide dismutase (SOD1) and withdrawal of trophic support (serum/nerve growth factor). Our previous results indicated that the initiating causes of death are different in each paradigm. However, bcl-2 rescues cells in either paradigm, suggesting common downstream elements to the cell death pathway. To determine whether the ICE [interleukin 1beta converting enzyme] family of proteases, which is required for apoptosis on trophic factor withdrawal, is also required for apoptosis induced by oxidative stress, we have developed a novel peptide inhibitor that mimics the common catalytic site of these enzymes and thereby blocks their access to substrates. This differs from the more usual pseudosubstrate approach to enzyme inhibition. Blockade of ICE family proteases by either this inhibitor or by a permeant competitive ICE family antagonist rescues PC12 cells from apoptotic death following apoptosis induced by down-regulation of SOD1, as well as from trophic factor/nerve growth factor deprivation. SOD1 down-regulation results in an increase in interleukin 1beta (IL- 1beta) production by the cells, and cell death under these conditions can be prevented by either blocking antibodies against IL-1beta or the IL-1 receptor antagonist (IL-1Ralpha). In contrast, trophic factor withdrawal does not increase IL-1beta secretion, and the blocking antibody failed to protect PC12 cells from trophic factor withdrawal, whereas the receptor antagonist was only partially protective at very high concentrations. There were substantial differences in the concentrations of pseudosubstrate inhibitors which rescued cells from SOD1 down-regulation and trophic factor deprivation. These results suggest the involvement of different members of the ICE family, different substrates, or both in the two different initiating causes of cell death.

The role of cysteine proteases in hypoxia-induced rat renal proximal tubular injury.

The role of the lysosomal proteases cathepsins B and L and the calcium-dependent cytosolic protease calpain in hypoxia-induced renal proximal tubular injury was investigated. As compared to normoxic tubules, cathepsin B and L activity, evaluated by the specific fluorescent substrate benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin, was not increased in hypoxic tubules or the medium used for incubation of hypoxic tubules in spite of high lactate dehydrogenase (LDH) release into the medium during hypoxia. These data in rat proximal tubules suggest that cathepsins are not released from lysosomes and do not gain access to the medium during hypoxia. An assay for calpain activity in isolated proximal tubules using the fluorescent substrate N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin was developed. The calcium ionophore ionomycin induced a dose-dependent increase in calpain activity. This increase in calpain activity occurred prior to cell membrane damage as assessed by LDH release. Tubular calpain activity increased significantly by 7.5 min of hypoxia, before there was significant LDH release, and further increased during 20 min of hypoxia. The cysteine protease inhibitor N-benzyloxycarbonyl-Val-Phe methyl ester (CBZ) markedly decreased LDH release after 20 min of hypoxia and completely prevented the increase in calpain activity during hypoxia. The increase in calpain activity during hypoxia and the inhibitor studies with CBZ therefore supported a role for calpain as a mediator of hypoxia-induced proximal tubular injury.

A molecular sensor system based on genetically engineered alkaline phosphatase.

Binding and signaling proteins based on Escherichia coli alkaline phosphatase (AP; EC 3.1.3.1) were designed for the detection of antibodies. Hybrid proteins were constructed by using wild-type AP and point mutants of AP [Asp-101 --> Ser (D101S) and Asp-153 --> Gly (D153G)]. The binding function of the hybrid proteins is provided by a peptide epitope inserted between amino acids 407 and 408 in AP. Binding of anti-epitope antibodies to the hybrid proteins modulates the enzyme activity of the hybrids; upon antibody binding, enzyme activity can increase to as much as 300% of the level of activity in the absence of antibody or can decrease as much as 40%, depending on the presence or absence of the point mutations in AP. The fact that modulation is altered from inhibition to activation by single amino acid changes in the active site of AP suggests that the mechanism for modulation is due to structural alterations upon antibody binding. Modulation is a general phenomenon. The properties of the system are demonstrated by using two epitopes, one from the V3 loop of human immunodeficiency virus type 1 gp120 protein and one from hepatitis C virus core protein, and corresponding monoclonal antibodies. The trend of modulation is consistent for all hybrids; those in wild-type AP are inhibited by antibody, while those in the AP mutants are activated by antibody. This demonstrates that modulation of enzyme activity of the AP-epitope hybrid proteins is not specific to either a particular epitope sequence or a particular antibody-epitope combination.

Protease footprinting reveals a surface on transcription factor TFIIB that serves as an interface for activators and coactivators.

Transcriptional stimulation by the model activator GAL4-VP16 (a chimeric protein consisting of the DNA-binding domain of the yeast activator GAL4 and the acidic activation domain of the herpes simplex virus protein VP16) involves a series of poorly understood protein-protein interactions between the VP16 activation domain and components of the RNA polymerase II general transcription machinery. One of these interactions is the VP16-mediated binding and recruitment of transcription factor TFIIB. However, TATA box-binding protein (TBP)-associated factors (TAFs), or coactivators, are required for this interaction to culminate in productive transcription complex assembly, and one such TAF, Drosophila TAF40, reportedly forms a ternary complex with VP16 and TFIIB. Due to TFIIB's central role in gene activation, we sought to directly visualize the surfaces of this protein that mediate formation of the ternary complex. We developed an approach called protease footprinting in which the broad-specificity proteases chymotrypsin and alkaline protease were used to probe binding of 32P-end-labeled TFIIB to GAL4-VP16 or TAF40. Analysis of the cleavage products revealed two regions of TFIIB protected by VP16 from protease attack, one of which overlapped with a region protected by TAF40. The close proximity of the VP16 and TAF40 binding sites on the surface of TFIIB suggests that this region could act as a regulatory interface mediating the effects of activators and coactivators on transcription complex assembly.