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.

Inhibition of cyclin D-CDK4/CDK6 activity is associated with an E2F-mediated induction of cyclin kinase inhibitor activity.

Alterations of various components of the cell cycle regulatory machinery that controls the progression of cells from a quiescent to a growing state contribute to the development of many human cancers. Such alterations include the deregulated expression of G1 cyclins, the loss of function of activities such as those of protein p16INK4a that control G1 cyclin-dependent kinase activity, and the loss of function of the retinoblastoma protein (RB), which is normally regulated by the G1 cyclin-dependent kinases. Various studies have revealed an inverse relationship in the expression of p16INK4a protein and the presence of functional RB in many cell lines. In this study we show that p16INK4a is expressed in cervical cancer cell lines in which the RB gene, Rb, is not functional, either as a consequence of Rb mutation or expression of the human papillomavirus E7 protein. We also demonstrate that p16INK4a levels are increased in primary cells in which RB has been inactivated by DNA tumor virus proteins. Given the role of RB in controlling E2F transcription factor activity, we investigated the role of E2F in controlling p16INK4a expression. We found that E2F1 overexpression leads to an inhibition of cyclin D1-dependent kinase activity and induces the expression of a p16-related transcript. We conclude that the accumulation of G1 cyclin-dependent kinase activity during normal G1 progression leads to E2F accumulation through the inactivation of RB, and that this then leads to the induction of cyclin kinase inhibitor activity and a shutdown of G1 kinase activity.

Protein-tyrosine phosphatase activity regulates osteoclast formation and function: inhibition by alendronate.

Alendronate (ALN), an aminobisphosphonate used in the treatment of osteoporosis, is a potent inhibitor of bone resorption. Its molecular target is still unknown. This study examines the effects of ALN on the activity of osteoclast protein-tyrosine phosphatase (PTP; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), called PTPepsilon. Using osteoclast-like cells generated by coculturing mouse bone marrow cells with mouse calvaria osteoblasts, we found by molecular cloning and RNA blot hybridization that PTPepsilon is highly expressed in osteoclastic cells. A purified fusion protein of PTPepsilon expressed in bacteria was inhibited by ALN with an IC50 of 2 microM. Other PTP inhibitors--orthovanadate and phenylarsine oxide (PAO)-inhibited PTPepsilon with IC50 values of 0.3 microM and 18 microM, respectively. ALN and another bisphosphonate, etidronate, also inhibited the activities of other bacterially expressed PTPs such as PTPsigma and CD45 (also called leukocyte common antigen). The PTP inhibitors ALN, orthovanadate, and PAO suppressed in vitro formation of multinucleated osteoclasts from osteoclast precursors and in vitro bone resorption by isolated rat osteoclasts (pit formation) with estimated IC50 values of 10 microM, 3 microM, and 0.05 microM, respectively. These findings suggest that tyrosine phosphatase activity plays an important role in osteoclast formation and function and is a putative molecular target of bisphosphonate action.

Phosphinate analogs of D-, D-dipeptides: slow-binding inhibition and proteolysis protection of VanX, a D-, D-dipeptidase required for vancomycin resistance in Enterococcus faecium.

VanX is a D-Ala-D-Ala dipeptidase that is essential for vancomycin resistance in Enterococcus faecium. Contrary to most proteases and peptidases, it prefers to hydrolyze the amino substrate but not the related kinetically and thermodynamically more favorable ester substrate D-Ala-D-lactate. The enzymatic activity of VanX was previously found to be inhibited by the phosphinate analogs of the proposed tetrahedral intermediate for hydrolysis of D-Ala-D-Ala. Here we report that such phosphinates are slow-binding inhibitors. D-3-[(1-Aminoethyl)phosphinyl]-D-2-methylpropionic acid I showed a time-dependent onset of inhibition of VanX and a time-dependent return to uninhibited steady-state rates upon dilution of the enzyme/inhibitor mixture. The initial inhibition constant Ki after immediate addition of VanX to phosphinate I to form the E-I complex is 1.5 microM but is then lowered by a relatively slow isomerization step to a second complex, E-I*, with a final K*i of 0.47 microM. This slow-binding inhibition reflects a Km/K*i ratio of 2900:1. The rate constant for the slow dissociation of complex E-I* is 0.24 min-1. A phosphinate analog with an ethyl group replacing what would be the side chain of the second D-alanyl residue in the normal tetrahedral adduct gives a K*i value of 90 nM. Partial proteolysis of VanX reveals two protease-sensitive loop regions that are protected by the intermediate analog phosphinate, indicating that they may be part of the VanX active site.

Trans-dominant inhibitory human immunodeficiency virus type 1 protease monomers prevent protease activation and virion maturation.

Production of infectious human immunodeficiency virus (HIV) requires proper polyprotein processing by the dimeric viral protease. The trans-dominant inhibitory activity of a defective protease monomer with the active site Asp-25 changed to Asn was measured by transient transfection. A proviral plasmid that included the drug-selectable Escherichia coli gpt gene was used to deliver the wild-type (wt) or mutant proteases to cultured cells. Coexpression of the wt proviral DNA (HIV-gpt) with increasing amounts of the mutant proviral DNA (HIV-gpt D25N) results in a concomitant decrease in proteolytic activity monitored by in vivo viral polyprotein processing. The viral particles resulting from inactivation of the protease were mostly immature, consisting predominantly of unprocessed p55gag and p160gag-pol polyproteins. In the presence of HIV-1 gp160 env, the number of secreted noninfectious particles correlated with the presence of increasing amounts of the defective protease. Greater than 97% reduction in infectivity was observed at a 1:6 ratio of wt to defective protease DNA. This provides an estimate of the level of inhibition required for effectively preventing virion processing. Stable expression of the defective protease in monkey cells reduced the yield of infectious particles from these cells by 90% upon transfection with the wt proviral DNA. These results show that defective subunits of the viral protease exert a trans-dominant inhibitory effect resulting from the formation of catalytically compromised heterodimers in vivo, ultimately yielding noninfectious viral particles.

Inhibition of phosphatidylinositol 3-kinase activity by association with 14-3-3 proteins in T cells.

Proteins of the 14-3-3 family can associate with, and/or modulate the activity of, several protooncogene and oncogene products and, thus, are implicated in regulation of signaling pathways. We report that 14-3-3 is associated with another important transducing enzyme, phosphatidylinositol 3-kinase (PI3-K). A recombinant 14-3-3 fusion protein bound several tyrosine-phosphorylated proteins from antigen receptor-stimulated T lymphocytes. PI3-K was identified by immunoblotting and enzymatic assays as one of the 14-3-3-binding proteins in resting or activated cells. Moreover, endogenous 14-3-3 and PI3-K were coimmunoprecipitated from intact T cells. Far-Western blots of gel-purified, immunoprecipitated PI3-K with a recombinant 14-3-3 fusion protein revealed direct binding of 14-3-3 to the catalytic subunit (p110) of PI3-K. Finally, anti-phosphotyrosine immunoprecipitates from activated, 14-3-3-overexpressing cells contained lower PI3-K enzymatic activity than similar immunoprecipitates from control cells. These findings suggest that association of 14-3-3 with PI3-K in hematopoietic (and possibly other) cells regulates the enzymatic activity of PI3-K during receptor-initiated signal transduction.

Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition.

Tobacco plants were transformed with a cDNA clone of chymotrypsin/trypsin-specific potato proteinase inhibitor II (PI2) under the control of a constitutive promoter. Although considerable levels of transgene expression could be demonstrated, the growth of Spodoptera exigua larvae fed with detached leaves of PI2-expressing plants was not affected. Analysis of the composition of tryptic gut activity demonstrated that only 18% of the proteinase activity of insects reared on these transgenic plants was sensitive to inhibition by PI2, whereas 78% was sensitive in insects reared on control plants. Larvae had compensated for this loss of tryptic activity by a 2.5-fold induction of new activity that was insensitive to inhibition by PI2. PI2-insensitive proteolytic activity was also induced in response to endogenous proteinase inhibitors of tobacco; therefore, induction of such proteinase activity may represent the mechanism by which insects that feed on plants overcome plant proteinase inhibitor defense.

Estradiol and the inhibition of hypothalamic gonadotropin-releasing hormone pulse generator activity in the rhesus monkey.

In mammals, gonadal function is controlled by a hypothalamic signal generator that directs the pulsatile release of gonadotropin-releasing hormone (GnRH) and the consequent pulsatile secretion of luteinizing hormone. In female rhesus monkeys, the electrophysiological correlates of GnRH pulse generator activity are abrupt, rhythmic increases in hypothalamic multiunit activity (MUA volleys), which represent the simultaneous increase in firing rate of individual neurons. MUA volleys are arrested by estradiol, either spontaneously at midcycle or after the administration of the steroid. Multiunit recordings, however, provide only a measure of total neuronal activity, leaving the behavior of the individual cells obscure. This study was conducted to determine the mode of action of estradiol at the level of single neurons associated with the GnRH pulse generator. Twenty-three such single units were identified by cluster analysis of multiunit recordings obtained from a total of six electrodes implanted in the mediobasal hypothalamus of three ovariectomized rhesus monkeys, and their activity was monitored before and after estradiol administration. The bursting of all 23 units was arrested within 4 h of estradiol administration although their baseline activity was maintained. The bursts of most units reappeared at the same time as the MUA volleys, the recovery of some was delayed, and one remained inhibited for the duration of the study (43 days). The results indicate that estradiol does not desynchronize the bursting of single units associated with the GnRH pulse generator but that it inhibits this phenomenon. The site and mechanism of action of estradiol in this regard remain to be determined.

Antitumor promotion by phenolic antioxidants: inhibition of AP-1 activity through induction of Fra expression.

Induction of phase 2 detoxification enzymes by phenolic antioxidants can account for prevention of tumor initiation but cannot explain why these compounds inhibit tumor promotion. Phase 2 genes are induced through an antioxidant response element (ARE). Although the ARE resembles an AP-1 binding site, we show that the major ARE binding and activating protein is not AP-1. Interestingly, AP-1 DNA binding activity was induced by the phenolic antioxidant tert-butylhydroquinone (BHQ), but the induction of AP-1 transcriptional activity by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) was inhibited by this compound. BHQ induced expression of c-jun, junB, fra-1, and fra-2, which encode AP-1 components, but was a poor inducer of c-fos and had no effect on fosB. Like c-Fos and FosB, the Fra proteins heterodimerize with Jun proteins to form stable AP-1 complexes. However, Fra-containing AP-1 complexes have low transactivation potential. Furthermore, Fra-1 repressed AP-1 activity induced by either TPA or expression of c-Jun and c-Fos. We therefore conclude that inhibitory AP-1 complexes composed of Jun-Fra heterodimers, induced by BHQ, antagonize the transcriptional effects of the tumor promoter TPA, which are mediated by Jun-Fos heterodimers. Since AP-1 is an important mediator of tumor promoter action, these findings may explain the anti-tumor-promoting activity of phenolic antioxidants.

Profound activity of the anti-cancer drug bortezomib against Echinococcus multilocularis metacestodes identifies the proteasome as a novel drug target for cestodes.

A library of 426 FDA-approved drugs was screened for in vitro activity against E. multilocularis metacestodes employing the phosphoglucose isomerase (PGI) assay. Initial screening at 20 µM revealed that 7 drugs induced considerable metacestode damage, and further dose-response studies revealed that bortezomib (BTZ), a proteasome inhibitor developed for the chemotherapy of myeloma, displayed high anti-metacestodal activity with an EC50 of 0.6 µM. BTZ treatment of E. multilocularis metacestodes led to an accumulation of ubiquinated proteins and unequivocally parasite death. In-gel zymography assays using E. multilocularis extracts demonstrated BTZ-mediated inhibition of protease activity in a band of approximately 23 kDa, the same size at which the proteasome subunit beta 5 of E. multilocularis could be detected by Western blot. Balb/c mice experimentally infected with E. multilocularis metacestodes were used to assess BTZ treatment, starting at 6 weeks post-infection by intraperitoneal injection of BTZ. This treatment led to reduced parasite weight, but to a degree that was not statistically significant, and it induced adverse effects such as diarrhea and neurological symptoms. In conclusion, the proteasome was identified as a drug target in E. multilocularis metacestodes that can be efficiently inhibited by BTZ in vitro. However, translation of these findings into in vivo efficacy requires further adjustments of treatment regimens using BTZ, or possibly other proteasome inhibitors.

Cloning and functional expression of venom prothrombin activator protease from Pseudonaja textilis with whole blood procoagulant activity

The snake venom group C prothrombin activators contain a number of components that enhance the rate of prothrombin activation. The cloning and expression of full-length cDNA for one of these components, an activated factor X (factor Xa)-like protease from Pseudonaja textilis as well as the generation of functional chimeric constructs with procoagulant activity were described. The complete cDNA codes for a propeptide, light chain, activation peptide (AP) and heavy chain related in sequence to mammalian factor X. Efficient expression of the protease was achieved with constructs where the AP was deleted and the cleavage sites between the heavy and light chains modified, or where the AP was replaced with a peptide involved in insulin receptor processing. In human kidney cells (H293F) transfected with these constructs, up to 80% of the pro-form was processed to heavy and light chains. Binding of the protease to barium citrate and use of specific antibodies demonstrated that gamma-carboxylation of glutamic acid residues had occurred on the light chain in both cases, as observed in human factor Xa and the native P. textilis protease. The recombinant protease caused efficient coagulation of whole citrated blood and citrated plasma that was enhanced by the presence of Ca2+. This study identified the complete cDNA sequence of a factor Xa-like protease from P. textilis and demonstrated for the first time the expression of a recombinant form of P. textilis protease capable of blood coagulation.

Potencies of human immunodeficiency virus protease inhibitors in vitro against Plasmodium falciparum and in vivo against murine malaria

Parasite resistance to antimalarial drugs is a serious threat to human health, and novel agents that act on enzymes essential for parasite metabolism, such as proteases, are attractive targets for drug development. Recent studies have shown that clinically utilized human immunodeficiency virus (HIV) protease inhibitors can inhibit the in vitro growth of Plasmodium falciparum at or below concentrations found in human plasma after oral drug administration. The most potent in vitro antimalarial effects have been obtained for parasites treated with saquinavir, ritonavir, or lopinavir, findings confirmed in this study for a genetically distinct P. falciparum line (3D7). To investigate the potential in vivo activity of antiretroviral protease inhibitors (ARPIs) against malaria, we examined the effect of ARPI combinations in a murine model of malaria. In mice infected with Plasmodium chabaudi AS and treated orally with ritonavir-saquinavir or ritonavir-lopinavir, a delay in patency and a significant attenuation of parasitemia were observed. Using modeling and ligand docking studies we examined putative ligand binding sites of ARPIs in aspartyl proteases of P. falciparum (plasmepsins II and IV) and P. chabaudi (plasmepsin) and found that these in silico analyses support the antimalarial activity hypothesized to be mediated through inhibition of these enzymes. In addition, in vitro enzyme assays demonstrated that P. falciparum plasmepsins II and IV are both inhibited by the ARPIs saquinavir, ritonavir, and lopinavir. The combined results suggest that ARPIs have useful antimalarial activity that may be especially relevant in geographical regions where HIV and P. falciparum infections are both endemic.

Sugarcane ShSUT1: analysis of sucrose transport activity and inhibition by sucralose

Plant sucrose transporters (SUTs) are members of the glycoside-pentoside-hexuronide (GPH) cation symporter family (TC2.A.2) that is part of the major facilitator superfamily (MFS). All plant SUTs characterized to date function as proton-coupled symporters and catalyze the cellular uptake of sucrose. SUTs are involved in loading sucrose into the phloem and sink tissues, such as seeds, roots and flowers. Because monocots are agriculturally important, SUTs from cereals have been the focus of recent research. Here we present a functional analysis of the SUT ShSUT1 from sugarcane, an important crop species grown for its ability to accumulate high amounts of sucrose in the stem. ShSUT1 was previously shown to be expressed in maturing stems and plays an important role in the accumulation of sucrose in this tissue. Using two-electrode voltage clamping in Xenopus oocytes expressing ShSUT1, we found that ShSUT1 is highly selective for sucrose, but has a relatively low affinity for sucrose (K-0.5 = 8.26 mM at pH 5.6 and a membrane potential of -137 mV). We also found that the sucrose analog sucralose (4,1 ',6 '-trichloro-4,1 ',6 '-trideoxygalactosucrose) is a competitive inhibitor of ShSUT1 with an inhibition coefficient (K-i) of 16.5 mM. The presented data contribute to our understanding of sucrose transport in plants in general and in monocots in particular.

Inhibition of transforming growth factor-beta 1 signaling attenuates ataxia telanglectasia mutated activity in response to genotoxic stress

Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor beta (TGF beta)-1, which is activated by radiation, is a potent and pleiotropic mediator of physiologic and pathologic processes. Here we show that TGF beta inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgf beta 1 nail murine epithelial cells or human epithelial cells treated with a small-molecule inhibitor of TGF beta type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17, and p53; reduced gamma H2AX radiation-induced foci; and increased radiosensitivity compared with TGF beta competent cells. We determined that loss of TGF beta signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGF beta restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM, which directs epithelial cell stress responses, cell fate, and tissue integrity. Thus, Tgf beta 1, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGF beta may be used to advantage in cancer therapy.