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.

An isoleucine/leucine residue in the carboxyltransferase domain of acetyl-CoA carboxylase is critical for interaction with aryloxyphenoxypropionate and cyclohexanedione inhibitors

cDNA fragments encoding the carboxyltransferase domain of the multidomain plastid acetyl-CoA carboxylase (ACCase) from herbicide-resistant maize and from herbicide-sensitive and herbicide-resistant Lolium rigidum were cloned and sequenced. A Leu residue was found in ACCases from herbicide-resistant plants at a position occupied by Ile in all ACCases from sensitive grasses studied so far. Leu is present at the equivalent position in herbicide-resistant ACCases from other eukaryotes. Chimeric ACCases containing a 1000-aa fragment of two ACCase isozymes found in a herbicide-resistant maize were expressed in a yeast ACC1 null mutant to test herbicide sensitivity of the enzyme in vivo and in vitro. One of the enzymes was resistant/tolerant, and one was sensitive to haloxyfop and sethoxydim, rendering the gene-replacement yeast strains resistant and sensitive to these compounds, respectively. The sensitive enzyme has an Ile residue, and the resistant one has a Leu residue at the putative herbicide-binding site. Additionally, a single Ile to Leu replacement at an equivalent position changes the wheat plastid ACCase from sensitive to resistant. The effect of the opposite substitution, Leu to Ile, makes Toxoplasma gondii apicoplast ACCase resistant to haloxyfop and clodinafop. In this case, inhibition of the carboxyltransferase activity of ACCase (second half-reaction) of a large fragment of the Toxoplasma enzyme expressed in Escherichia coli was tested. The critical amino acid residue is located close to a highly conserved motif of the carboxyltransferase domain, which is probably a part of the enzyme active site, providing the basis for the activity of fop and dim herbicides.

Nonnucleoside reverse transcriptase inhibitors are chemical enhancers of dimerization of the HIV type 1 reverse transcriptase

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are allosteric inhibitors of the HIV type 1 (HIV-1) reverse transcriptase (RT). Yeast grown in the presence of many of these drugs exhibited dramatically increased association of the p66 and p51 subunits of the HIV-1 RT as reported by a yeast two-hybrid assay. The enhancement required drug binding by RT; introduction of a drug-resistance mutation into the p66 construct negated the enhancement effect. The drugs could also induce heterodimerization of dimerization defective mutants. Coimmunoprecipitation of RT subunits from yeast lysates confirmed the induction of heterodimer formation by the drugs. In vitro-binding studies indicate that NNRTIs can bind tightly to p66 but not p51 and then mediate subsequent heterodimerization. This study demonstrates an unexpected effect of NNRTIs on the assembly of RT subunits.

γ-Secretase inhibitors repress thymocyte development

A major therapeutic target in the search for a cure to the devastating Alzheimer's disease is γ-secretase. This activity resides in a multiprotein enzyme complex responsible for the generation of Aβ42 peptides, precipitates of which are thought to cause the disease. γ-Secretase is also a critical component of the Notch signal transduction pathway; Notch signals regulate development and differentiation of adult self-renewing cells. This has led to the hypothesis that therapeutic inhibition of γ-secretase may interfere with Notch-related processes in adults, most alarmingly in hematopoiesis. Here, we show that application of γ-secretase inhibitors to fetal thymus organ cultures interferes with T cell development in a manner consistent with loss or reduction of Notch1 function. Progression from an immature CD4−/CD8− state to an intermediate CD4+/CD8+ double-positive state was repressed. Furthermore, treatment beginning later at the double-positive stage specifically inhibited CD8+ single-positive maturation but did not affect CD4+ single-positive cells. These results demonstrate that pharmacological γ-secretase inhibition recapitulates Notch1 loss in a vertebrate tissue and present a system in which rapid evaluation of γ-secretase-targeted pharmaceuticals for their ability to inhibit Notch activity can be performed in a relevant context.

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.

Dim-Red-Light-Induced Increase in Polar Auxin Transport in Cucumber Seedlings1 : I. Development of Altered Capacity, Velocity, and Response to Inhibitors

We have developed and characterized a system to analyze light effects on auxin transport independent of photosynthetic effects. Polar transport of [3H]indole-3-acetic acid through hypocotyl segments from etiolated cucumber (Cucumis sativus L.) seedlings was increased in seedlings grown in dim-red light (DRL) (0.5 μmol m−2 s−1) relative to seedlings grown in darkness. Both transport velocity and transport intensity (export rate) were increased by at least a factor of 2. Tissue formed in DRL completely acquired the higher transport capacity within 50 h, but tissue already differentiated in darkness acquired only a partial increase in transport capacity within 50 h of DRL, indicating a developmental window for light induction of commitment to changes in auxin transport. This light-induced change probably manifests itself by alteration of function of the auxin efflux carrier, as revealed using specific transport inhibitors. Relative to dark controls, DRL-grown seedlings were differentially less sensitive to two inhibitors of polar auxin transport, N-(naphth-1-yl) phthalamic acid and 2,3,5-triiodobenzoic acid. On the basis of these data, we propose that the auxin efflux carrier is a key target of light regulation during photomorphogenesis.

Chickpea Defensive Proteinase Inhibitors Can Be Inactivated by Podborer Gut Proteinases1

Developing chickpea (Cicer arietinum L.) seeds 12 to 60 d after flowering (DAF) were analyzed for proteinase inhibitor (Pi) activity. In addition, the electrophoretic profiles of trypsin inhibitor (Ti) accumulation were determined using a gel-radiographic film-contact print method. There was a progressive increase in Pi activity throughout seed development, whereas the synthesis of other proteins was low from 12 to 36 DAF and increased from 36 to 60 DAF. Seven different Ti bands were present in seeds at 36 DAF, the time of maximum podborer (Helicoverpa armigera) attack. Chickpea Pis showed differential inhibitory activity against trypsin, chymotrypsin, H. armigera gut proteinases, and bacterial proteinase(s). In vitro proteolysis of chickpea Ti-1 with various proteinases generated Ti-5 as the major fragment, whereas Ti-6 and -7 were not produced. The amount of Pi activity increased severalfold when seeds were injured by H. armigera feeding. In vitro and in vivo proteolysis of the early- and late-stage-specific Tis indicated that the chickpea Pis were prone to proteolytic digestion by H. armigera gut proteinases. These data suggest that survival of H. armigera on chickpea may result from the production of inhibitor-insensitive proteinases and by secretion of proteinases that digest chickpea Pis.

TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors

To achieve an efficient intracellular drug and DNA delivery, attempts were made to target microparticulate drug carriers into cytoplasm bypassing the endocytotic pathway. TAT peptides derived from the HIV-1 TAT protein facilitate intracellular delivery of proteins and small colloidal particles. We demonstrated that relatively large drug carriers, such as 200-nm liposomes, can also be delivered into cells by TAT peptide attached to the liposome surface. Liposomes were fluorescently labeled with membranotropic rhodamine-phosphatidylethanolamine or by entrapping FITC-dextran. Incubation of fluorescent TAT liposomes with mouse Lewis lung carcinoma cells, human breast tumor BT20 cells, and rat cardiac myocyte H9C2 results in intracellular localization of certain liposomes. Steric hindrances for TAT peptide⋅cell interaction (attachment of TAT directly to the liposome surface without spacer or the presence of a high MW polyethylene glycol on the liposome surface) abolish liposome internalization, evidencing the importance of direct contact of TAT peptide with the cell surface. Low temperature or metabolic inhibitors, sodium azide or iodoacetamide, have little influence on the translocation of TAT liposomes into cells, confirming the energy-independent character of this process. The approach may have important implications for drug delivery directly into cell cytoplasm.

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.

A genetic selection for Caenorhabditis elegans synaptic transmission mutants.

We have isolated 165 Caenorhabditis elegans mutants, representing 21 genes, that are resistant to inhibitors of cholinesterase (Ric mutants). Since mutations in 20 of the genes appear not to affect acetylcholine reception, we suggest that reduced acetylcholine release contributes to the Ric phenotype of most Ric mutants. Mutations in 15 of the genes lead to defects in a gamma-aminobutyric acid-dependent behavior; these genes are likely to encode proteins with general, rather than cholinergic-specific, roles in synaptic transmission. Ten of the genes have been cloned. Seven encode homologs of proteins that function in the synaptic vesicle cycle: two encode cholinergic-specific proteins, while five encode general presynaptic proteins. Two other Ric genes encode homologs of G-protein signaling molecules. Our assessment of synaptic function in Ric mutants, combined with the homologies of some Ric mutants to presynaptic proteins, suggests that the analysis of Ric genes will continue to yield insights into the regulation and functioning of synapses.

Identification of metabolic pathways of brain angiotensin II and III using specific aminopeptidase inhibitors: predominant role of angiotensin III in the control of vasopressin release.

Angiotensin (Ang) II and Ang III are two peptide effectors of the brain renin-angiotensin system that participate in the control of blood pressure and increase water consumption and vasopressin release. In an attempt to delineate the respective roles of these peptides in the regulation of vasopressin secretion, their metabolic pathways and their effects on vasopressin release were identified in vivo. For this purpose, we used recently developed selective inhibitors of aminopeptidase A (APA) and aminopeptidase N (APN), two enzymes that are believed to be responsible for the N-terminal cleavage of Ang II and Ang III, respectively. Mice received [3H]Ang II intracerebroventricularly (i.c.v.) in the presence or absence of the APN inhibitor, EC33 (3-amino-4-thio-butyl sulfonate) of the APN inhibitor, EC27 (2-amino-pentan-1,5-dithiol). [3H]Ang II and [3H]Ang III levels were evaluated from hypothalamus homogenates by HPLC. EC33 increased the half-life of [3H]Ang II 2.6-fold and completely blocked the formation of [3H]Ang III, whereas EC27 increased the half-life of [3H]Ang III 2.3-fold. In addition, the effects of EC33 and EC27 on Ang-induced vasopressin release were studied in mice. Ang II was injected i.c.v. in the presence or absence of EC33, and plasma vasopressin levels were estimated by RIA. While vasopressin levels were increased 2-fold by Ang II (5 ng), EC33 inhibited Ang II-induced vasopressin release in a dose-dependent manner. In contrast, EC27 injected alone increased in a dose-dependent manner vasopressin levels. The EC27-induced vasopressin release was completely blocked by the coadministration of the Ang receptor antagonist (Sar1-Ala8) Ang II. These results demonstrate for the first time that (i) APA and APN are involved in vivo in the metabolism of brain Ang II and Ang III, respectively, and that (ii) the action of Ang II on vasopressin release depends upon the prior conversion of Ang II to Ang III. This shows that Ang III behaves as one of the main effector peptides of the brain renin-angiotensin system in the control of vasopressin release.

Menstrual breakdown of human endometrium can be mimicked in vitro and is selectively and reversibly blocked by inhibitors of matrix metalloproteinases.

The mechanisms underlying the menstrual lysis leading to shedding of the human endometrium and its accompanying bleeding are still largely unknown. In particular, whether breakdown of the endometrial fibrillar extra-cellular matrix that precedes bleeding depends on aspartic-, cysteine-, serine-, or metalloproteinases remains unclear. In the present study, menstrual regression of the human endometrium was mimicked in organ culture. Whereas sex steroids could preserve tissue integrity only in nonperimenstrual explants, matrix breakdown upon sex steroid deprivation was completely and reversibly inhibited at all stages of the menstrual cycle by specific inhibitors of matrix metalloproteinases, but not by inhibitors of the other classes of proteinases. Matrix metalloproteinases are thus identified as the key class of proteinases involved in the initiation of menstruation.

Structural basis for selectivity of the isoquinoline sulfonamide family of protein kinase inhibitors.

A large family of isoquinoline sulfonamide compounds inhibits protein kinases by competing with adenosine triphosphates(ATP), yet interferes little with the activity of other ATP-using enzymes such as ATPases and adenylate cyclases. One such compound, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide (CK17), is selective for casein kinase-1 isolated from a variety of sources. Here we report the crystal structure of the catalytic domain of Schizosaccharomyces pombe casein kinase-1 complexed with CK17, refined to a crystallographic R-factor of 17.8% at 2.5 angstrom resolution. The structure provides new insights into the mechanism of the ATP-competing inhibition and the origin of their selectivity toward different protein kinases. Selectivity for protein kinases versus other enzymes is achieved by hydrophobic contacts and the hydrogen bond with isoquinoline ring. We propose that the hydrogen bond involving the ring nitrogen-2 atom of the isoquinoline must be preserved, but that the ring can flip depending on the chemical substituents at ring positions 5 and 8. Selectivity for individual members of the protein kinase family is achieved primarily by interactions with these substituents.

Inhibitors of HIV-1 replication that inhibit HIV integrase.

HIV-1 replication depends on the viral enzyme integrase that mediates integration of a DNA copy of the virus into the host cell genome. This enzyme represents a novel target to which antiviral agents might be directed. Three compounds, 3,5-dicaffeoylquinic acid, 1-methoxyoxalyl-3,5-dicaffeoylquinic acid, and L-chicoric acid, inhibit HIV-1 integrase in biochemical assays at concentrations ranging from 0.06-0.66 microgram/ml; furthermore, these compounds inhibit HIV-1 replication in tissue culture at 1-4 microgram/ml. The toxic concentrations of these compounds are fully 100-fold greater than their antiviral concentrations. These compounds represent a potentially important new class of antiviral agents that may contribute to our understanding of the molecular mechanisms of viral integration. Thus, the dicaffeoylquinic acids are promising leads to new anti-HIV therapeutics and offer a significant advance in the search for new HIV enzyme targets as they are both specific for HIV-1 integrase and active against HIV-1 in tissue culture.

Activation and phosphorylation of a pleckstrin homology domain containing protein kinase (RAC-PK/PKB) promoted by serum and protein phosphatase inhibitors.

Treatment of quiescent Swiss 3T3 fibroblasts with serum, or with the phosphatase inhibitors okadaic acid and vanadate, induced a 2- to 11-fold activation of the serine/ threonine RAC protein kinase (RAC-PK). Kinase activation was accompanied by decreased mobility of RAC-PK on SDS/PAGE such that three electrophoretic species (a to c) of the kinase were detected by immunoblot analysis, indicative of differentially phosphorylated forms. Addition of vanadate to arrested cells increased the RAC-PK phosphorylation level 3-to 4-fold. Unstimulated RAC-PK was phosphorylated predominantly on serine, whereas the activated kinase was phosphorylated on both serine and threonine residues. Treatment of RAC-PK in vitro with protein phosphatase 2A led to kinase inactivation and an increase in electrophoretic mobility. Deletion of the N-terminal region containing the pleckstrin homology domain did not affect RAC-PK activation by okadaic acid, but it reduced vanadate-stimulated activity and also blocked the serum-induced activation. Deletion of the serine/threonine rich C-terminal region impaired both RAC-PKalpha basal and vanadate-stimulated activity. Studies using a kinase-deficient mutant indicated that autophosphorylation is not involved in RAC-PKalpha activation. Stimulation of RAC-PK activity and electrophoretic mobility changes induced by serum were sensitive to wortmannin. Taken together the results suggest that RAC-PK is a component of a signaling pathway regulated by phosphatidylinositol (PI) 3-kinase, whose action is required for RAC-PK activation by phosphorylation.