The generation of immunogenic peptides can be selectively increased or decreased by proteolytic enzyme inhibitors

The ability of splenic APC and a B cell hybridoma (LS.102.9) to process and present OVA to a panel of T-T hybridomas with different fine specificities was investigated. Splenic APC process and present OVA to all the T-T hybrids. The B cell hybridoma could similarly process and present OVA to some T-T hybrids but was very inefficient in stimulating two of the T cell hybridomas. The presentation of native OVA to these two T-T hybrids was significantly increased by leupeptin. Pulsing experiments demonstrated that leupeptin acted on the APC at a step before the processed Ag was displayed on the cell surface in association with MHC molecules. Leupeptin has no effect on the presentation of OVA peptides by LS.102.9 to the T-T hybrids. Leupeptin inhibits the generation of the epitopes of OVA that LS.102.9 produces under basal conditions. We also surveyed the effect of other protease inhibitors and observed similar augmenting and inhibitory effects on the presentation of selected OVA epitopes. The augmentation of processing by a protease inhibitor indicates that in the lysosomal/endosomal compartment proteases have capacity to both generate and destroy immunogenic peptides. Our data suggest that protease inhibitors could potentially be used as immunomodulators and are discussed in terms of physiology of the lysosomal/endosomal compartment.     

The probability of obtaining complex kinetic curves for enzyme mechanisms with cubic terms in the pseudo-steady state rate equations

A number of details required for the classification of 3 : 3 double reciprocal plots are provided. It is shown that the ν(S) plot for a 3 : 3 function can have at most four inflexions and at most two inflexions adjacent to a turning point. Using this information, a classification of 3 : 3 ν(S) plots into ten main varieties with several subclasses is reported. The problem of defining the probability with which a given mechanism can give rise to specific curve shape features is considered. Applying this technique, the probability with which four simple enzyme mechanisms can give rise to 3 : 3 curve shapes is computed. It is shown that a 3 : 3 saturation function can have no turning points, at most two inflexions and at most one inflexion in double reciprocal space. The probability with which the available 3 : 3 shapes can arise is also computed. It is concluded that, with realistic values for rate constants, chemically reasonable enzyme mechanisms leading to rate equations of degree n : n can generate most of the kinetic profiles available to a rational function of degree n : n with positive coefficients. The probability of obtaining specific curve shapes is not so characteristic of the particular mechanism for 3:3 rate equations as it is for 2:2 rate equations. The probability of obtaining highly complex curves with several turning points or inflexions is rather lower for the enzyme mechanisms than with general 3 : 3 rational functions. There is a high probability that 3 : 3 mechanisms will generate kinetic curves that are geometrically similar to those possible for degree 2 : 2 but this is not so for binding isotherms. Hence differentiating 3 : 3 from 2 : 2 rate equations from experimental kinetic data is more likely to be successful by non-linear regression to the whole data set than by demonstrating a specific 3 : 3 feature. Binding curves, on the other hand, for three or more sites should give Scatchard plots with inflexions, features not possible with second degree equations which are conic sections in this space.     

Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denaturants

Conditioned medium from cultured bovine aortic endothelial cells contains an inactive plasminogen activator inhibitor (PAI). This latent PAI can be "activated" with denaturants. For example, less than 0.01 units/microliter of PAI activity was detected in untreated conditioned medium, but medium treated with sodium dodecyl sulfate (1.7 mM), guanidine HCl (4 M), urea (12 M) or KSCN (6 M) contained 0.9, 1.9, 0.8, and 0.5 units/microliter, respectively. This effect was dose-dependent with respect to the particular reagent used, and the same concentration of reagent which induced PAI activity also stimulated the ability of a component in conditioned medium to form sodium dodecyl sulfate-stable complexes with exogenously added plasminogen activators. Neither activity was stimulated by extensive dialysis or by treatment with NaCl (5 M), Na2SO4 (2.8 M), or dicetyl phosphate (0.1%). Analysis of treated and untreated conditioned medium by gel filtration revealed that the latent and active PAIs migrated with apparent Mr values of 30,000 and 50,000, respectively. Thus, "activation" is associated with an increase in the apparent Mr of the molecule. These observations suggest that activation does not result from the removal of either a small dialyzable component from the medium, or of a large Mr component that is bound to the latent PAI. Other possible mechanisms of activation are discussed. We recently isolated an active PAI from bovine endothelial cells (van Mourik, J.A., Lawrence, D.A., and Loskutoff, D.J. (1984) J. Biol. Chem. 259, 14914-14921). Monospecific antiserum to this active PAI selectivity immunoprecipitated the latent PAI from conditioned medium. These results indicate that the two PAIs are immunologically related and suggest that the latent form is converted into the active form by the sodium dodecyl sulfate present during the purification.     

Polyglucosan neurotoxicity caused by glycogen branching enzyme deficiency can be reversed by inhibition of glycogen synthase

Uncontrolled elongation of glycogen chains, not adequately balanced by their branching, leads to the formation of an insoluble, presumably neurotoxic, form of glycogen called polyglucosan. To test the suspected pathogenicity of polyglucosans in neurological glycogenoses, we have modeled the typical glycogenosis Adult Polyglucosan Body Disease (APBD) by suppressing glycogen branching enzyme 1 (GBE1, EC 2.4.1.18) expression using lentiviruses harboring short hairpin RNA (shRNA). GBE1 suppression in embryonic cortical neurons led to polyglucosan accumulation and associated apoptosis, which were reversible by rapamycin or starvation treatments. Further analysis revealed that rapamycin and starvation led to phosphorylation and inactivation of glycogen synthase (GS, EC 2.4.1.11), dephosphorylated and activated in the GBE1‐suppressed neurons. These protective effects of rapamycin and starvation were reversed by overexpression of phosphorylation site mutant GS only if its glycogen binding site was intact. While rapamycin and starvation induce autophagy, autophagic maturation was not required for their corrective effects, which prevailed even if autophagic flux was inhibited by vinblastine. Furthermore, polyglucosans were not observed in any compartment along the autophagic pathway. Our data suggest that glycogen branching enzyme repression in glycogenoses can cause pathogenic polyglucosan buildup, which might be corrected by GS inhibition.

     

The Toxoplasma glucan phosphatase TgLaforin utilizes a distinct functional mechanism that can be exploited by therapeutic inhibitors

Toxoplasma gondii is an intracellular parasite that generates amylopectin granules (AGs), a polysaccharide associated with bradyzoites that define chronic T. gondii infection. AGs are postulated to act as an essential energy storage molecule that enable bradyzoite persistence, transmission, and reactivation. Importantly, reactivation can result in the life-threatening symptoms of toxoplasmosis. T. gondii encodes glucan dikinase and glucan phosphatase enzymes that are homologous to the plant and animal enzymes involved in reversible glucan phosphorylation and which are required for efficient polysaccharide degradation and utilization. However, the structural determinants that regulate reversible glucan phosphorylation in T. gondii are unclear. Herein, we define key functional aspects of the T. gondii glucan phosphatase TgLaforin (TGME49_205290). We demonstrate that TgLaforin possesses an atypical split carbohydrate-binding-module domain. AlphaFold2 modeling combined with hydrogen–deuterium exchange mass spectrometry and differential scanning fluorimetry also demonstrate the unique structural dynamics of TgLaforin with regard to glucan binding. Moreover, we show that TgLaforin forms a dual specificity phosphatase domain–mediated dimer. Finally, the distinct properties of the glucan phosphatase catalytic domain were exploited to identify a small molecule inhibitor of TgLaforin catalytic activity. Together, these studies define a distinct mechanism of TgLaforin activity, opening up a new avenue of T. gondii bradyzoite biology as a therapeutic target.     

Cell death induced by novel procaspase-3 activators can be reduced by growth factors

Caspase-3 is known as the key executioner caspase, activated in both the intrinsic and extrinsic apoptotic pathway, and an effector far downstream in the apoptotic cascade. Procaspase-activating compound-1 (PAC-1) and 1541 were launched as direct activators of procaspase-3 to caspase-3, and anticipated to be promising therapeutic agents for the treatment of cancer. PAC-1 has recently been evaluated in a phase I preclinical trial. However, little is known about the effect of these substances in cells. Activation of caspase-3 in whole cells may be more complicated than thought, as it is likely that this key protease is tightly regulated both in development and apoptosis. In this study, we investigated the effect of epidermal growth factor (EGF) on PAC-1-induced caspase-3 activity and cell death. We show that EGF can block caspase-3 activity generated by PAC-1, and protect both PC12 cells and primary cerebellar granule neurons against PAC-1-induced death. Similar results were obtained with 1541. Both substances reduced cellular p-ERK levels. Crosstalk between caspase-3 and growth factor signaling pathways may present a challenge for the use of such caspase-3-activating substances in cancer therapy, since aberrant growth factor signaling is frequently seen in malignant cells. This study adds important knowledge about cellular effects of procaspase-3 activators like PAC-1 and 1541. Effects mediated by these substances may also contribute to the understanding of caspase signaling in cells.     

The delta-endotoxin proteins accumulate in Escherichia coli as a protein-DNA complex that can be dissociated by hydrophobic interaction chromatography

The insecticidal protein CryIAc accumulated to form inclusion bodies in Escherichia coli upon overexpression of the cloned gene. The solubilized inclusion bodies contained the delta-endotoxin in association with DNA fragments of about 25 kb. The protein-DNA complex could be dissociated and the delta-endotoxin purified by hydrophobic interaction chromatography on phenyl-Sepharose. The DNA was washed out in the high-salt buffer while the delta-endotoxin was bound to the matrix and was eluted at 4 degrees C by a stepwise decreasing potassium chloride gradient. The DNA-protein complex also contained plasmids harbored by the host strain. The plasmid DNA associated with the complex became competent to transform E. coli only after it was dissociated from the delta-endotoxin. The hydrophobic interaction chromatography provides an efficient method for the purification of DNA-free activated toxin.     

The Trypanosoma cruzi enzyme TcGPXI is a glycosomal peroxidase and can be linked to trypanothione reduction by glutathione or tryparedoxin

Trypanosoma cruzi glutathione-dependent peroxidase I (TcGPXI) can reduce fatty acid, phospholipid, and short chain organic hydroperoxides utilizing a novel redox cycle in which enzyme activity is linked to the reduction of trypanothione, a parasite-specific thiol, by glutathione. Here we show that TcGPXI activity can also be linked to trypanothione reduction by an alternative pathway involving the thioredoxin-like protein tryparedoxin. The presence of this new pathway was first detected using dialyzed soluble fractions of parasite extract. Tryparedoxin was identified as the intermediate molecule following purification, sequence analysis, antibody studies, and reconstitution of the redox cycle in vitro. The system can be readily saturated by trypanothione, the rate-limiting step being the interaction of trypanothione with the tryparedoxin. Both tryparedoxin and TcGPXI operate by a ping-pong mechanism. Overexpression of TcGPXI in transfected parasites confers increased resistance to exogenous hydroperoxides. TcGPXI contains a carboxyl-terminal tripeptide (ARI) that could act as a targeting signal for the glycosome, a kinetoplastid-specific organelle. Using immunofluorescence, tagged fluorescent proteins, and biochemical fractionation, we have demonstrated that TcGPXI is localized to both the glycosome and the cytosol. The ability of TcGPXI to use alternative electron donors may reflect their availability at the corresponding subcellular sites.     

Rac1-regulated endothelial radiation response stimulates extravasation and metastasis that can be blocked by HMG-CoA reductase inhibitors

Radiotherapy (RT) plays a key role in cancer treatment. Although the benefit of ionizing radiation (IR) is well established, some findings raise the possibility that irradiation of the primary tumor not only triggers a killing response but also increases the metastatic potential of surviving tumor cells. Here we addressed the question of whether irradiation of normal cells outside of the primary tumor augments metastasis by stimulating the extravasation of circulating tumor cells. We show that IR exposure of human endothelial cells (EC), tumor cells (TC) or both increases TC-EC adhesion in vitro. IR-stimulated TC-EC adhesion was blocked by the HMG-CoA reductase inhibitor lovastatin. Glycyrrhizic acid from liquorice root, which acts as a Sialyl-Lewis X mimetic drug, and the Rac1 inhibitor NSC23766 also reduced TC-EC adhesion. To examine the in vivo relevance of these findings, tumorigenic cells were injected into the tail vein of immunodeficient mice followed by total body irradiation (TBI). The data obtained show that TBI dramatically enhances tumor cell extravasation and lung metastasis. This pro-metastatic radiation effect was blocked by pre-treating mice with lovastatin, glycyrrhizic acid or NSC23766. TBI of mice prior to tumor cell transplantation also stimulated metastasis, which was again blocked by lovastatin. The data point to a pro-metastatic trans-effect of RT, which likely rests on the endothelial radiation response promoting the extravasation of circulating tumor cells. Administration of the widely used lipid-lowering drug lovastatin prior to irradiation counteracts this process, likely by suppressing Rac1-regulated E-selectin expression following irradiation. The data support the concern that radiation exposure might increase the extravasation of circulating tumor cells and recommend co-administration of lipid-lowering drugs to avoid this adverse effect of ionizing radiation.     

The yeast oligosaccharyltransferase complex can be replaced by STT3 from Leishmania major

The key step of protein N-glycosylation is catalyzed by the multimeric oligosaccharyltransferase complex (OST). Biochemical and genetic studies have revealed that OST from Saccharomyces cerevisiae consists of nine subunits: Wbp1, Swp1, Stt3, Ost1, Ost2, Ost3, Ost4, Ost5, and Ost6. With the exception of Stt3, assumed to contain the catalytic site, little is known about the function of other OST subunits. The existence of the OST complex is suggested to allow substrate specificity and efficient transfer, a close interaction with the translocon and the prevention of protein folding to ensure the efficient co-translational modification of proteins. However, in the recently completed genome of the trypanosomatid parasite Leishmania major STT3 (of which four paralogs exist, STT3-1, STT3-2, STT3-3, and STT3-4) is the only OST subunit that can be identified. Here we report that L.m.STT3 proteins, except STT3-3, are able to complement stt3 deficiency in yeast during vegetative growth, but only poorly during sporulation. By blue native electrophoresis we demonstrate that the L.mSTT3 is active mainly as a free, monomeric enzyme. In cell-free assays and also in vivo we find that L.mSTT3, expressed in yeast, has a broad specificity for nonglucosylated lipid-linked mannose-oligosaccharides, typical for several protists. But when incorporated into the OST complex, L.mSTT3 transfers also the common eukaryotic Glc(3)Man(9)GlcNAc(2)-PP-Dol donor. Finally, three L.m.STT3 paralogs were shown to complement not only stt3 but also ost1, ost2, wbp1, or swp1 mutants. Thus, STT3 from Leishmania can substitute for the whole OST complex.     

REG can be used to detect cerebrovascular alteration caused by alcoholism

Despite recent evidence of the beneficial effects of moderate alcohol consumption in arteriosclerosis prevention, the neurotoxic effects of alcohol abuse are well known. Our hypothesis was that uncontrolled alcohol consumption may cause cerebrovascular damage detectable by rheoencepholography (REG), a noninvasive bio-impedance technique for estimating cerebral blood flow. Test subjects were 48 alcoholic patients in Hungary; the control group consisted of 12 drug-addicted and depressed patients in Hungary and 13 healthy male subjects in the United States. Additional subgroups were formed according to smoking habits and average daily alcohol dose. REG was measured by a computer-based system, "Cerberus"; REG anacrotic time above 180 ms was considered pathological. ANOVA showed that daily alcohol consumption and smoking were significantly higher in alcoholics than in drug-addicted and depressed patients. Twelve alcoholics showed a pathological REG anacrotic time. Longer REG anacrotic time was correlated with higher daily alcohol consumption. In the alcoholic group, the steeper regression line of REG slope reflected the pathological impact of alcohol abuse. The healthy control sample showed a nearly identical slope for both REG and age. The correlation of increased REG anacrotic time and daily alcohol consumption supports the hypothesis that REG detects accelerated cerebrovascular aging (arteriosclerosis) in alcoholic subjects.     

The enzyme rhodanese can be reactivated after denaturation in guanidinium chloride

For the first time, the enzyme rhodanese has been refolded after denaturation in guanidinium chloride (GdmHCl). Renaturation was by either (a) direct dilution into the assay, (b) intermediate dilution into buffer, or (c) dialysis followed by concentration and centrifugation. Method (c) preferentially retained active enzyme whose specific activity was 1140 IU/mg, which fell to 898 IU/mg after 6 days. The specific activity of native enzyme is 710 IU/mg. Progress curves were linear for the dialyzed enzyme, and kinetic analysis showed it had the same Km for thiosulfate as the native enzyme, but apparently displayed a higher turnover number. Progress curves for denatured enzyme directly diluted into assay mix showed as many as three phases: a lag during which no product formed; a first order reactivation; and an apparently linear steady state. An induction period was determined by extrapolating the steady-state line to the time axis. The percent reactivation fell to 7% (t1/2 = 10 min) as the time increased between GdmHCl dilution and the start of the assay, independent of the presence of thiosulfate. The induction period, which decreased to zero as the incubation time increased, was retained in the presence of thiosulfate. There were no observable differences between native and renatured protein by electrophoresis or fluorescence spectroscopy. Previous reports of some refolding of urea-denatured rhodanese (Stellwagen, E. (1979) J. Mol. Biol. 135, 217-229) were confirmed, extended, and compared with results using GdmHCl. A working hypothesis is that rhodanese refolding involves intermediates that partition into active and inactive products. These intermediates may result from nucleation of the two rhodanese domains, which exposes hydrophobic surfaces that become the interdomain interface in the correctly folded protein.     

Simple enzyme kinetic mechanisms that can give all possible velocity profiles with chemically reasonable rate constant values

It is shown that all rate profiles and double reciprocal plots possible for arbitrary rational functions of degree n : n can be given by enzyme mechanisms in which rate constant values are limited by physical constraints. The significance of this finding in the theory of complex kinetics is explained, and the application in the area of non-linear regression analysis of complex kinetic data and model discrimination, is discussed.     

Cell surface colony-stimulating factor 1 can be cleaved by TNF-alpha converting enzyme or endocytosed in a clathrin-dependent manner

CSF-1 is a hemopoietic growth factor, which plays an essential role in macrophage and osteoclast development. Alternative splice variants of CSF-1 are synthesized as soluble or membrane-anchored molecules, although membrane CSF-1 (mCSF-1) can be cleaved from the cell membrane to become soluble CSF-1. The activities involved in this proteolytic processing, also referred to as ectodomain shedding, remain poorly characterized. In the present study, we examined the properties of the mCSF-1 sheddase in cell-based assays. Shedding of mCSF-1 was up-regulated by phorbol ester treatment and was inhibited by the metalloprotease inhibitors GM6001 and tissue inhibitor of metalloproteases 3. Moreover, the stimulated shedding of mCSF-1 was abrogated in fibroblasts lacking the TNF-alpha converting enzyme (TACE, also known as a disintegrin and metalloprotease 17) and was rescued by expression of wild-type TACE in these cells, strongly suggesting that the stimulated shedding is TACE dependent. Additionally, we observed that mCSF-1 is predominantly localized to intracellular membrane compartments and is efficiently internalized in a clathrin-dependent manner. These results indicate that the local availability of mCSF-1 is actively regulated by ectodomain shedding and endocytosis. This mechanism may have important implications for the development and survival of monocyte lineage cells.     

Gin mutants that can be suppressed by a Fis-independent mutation.

The Gin invertase of bacteriophage Mu mediates recombination between two inverted gix sites. Recombination requires the presence of a second protein, Fis, which binds to an enhancer sequence. We have isolated 24 different mutants of Gin that are impaired in DNA inversion but proficient in DNA binding. Six of these mutants could be suppressed for inversion by introduction of a second mutation, which when present in the wild-type gin gene causes a Fis-independent phenotype. Only one of the six resulting double mutants shows an inversion efficiency which is comparable to that of the wild-type Gin and which is independent of Fis. The corresponding mutation, M to I at position 108 (M108I), is located in a putative alpha-helical structure, which in the homologous gamma delta resolvase has been implicated in dimerization. The properties of the M108I mutant suggest that in Gin this dimerization helix might also be the target for Fis interaction. The five other mutants that show a restored inversion after introduction of a Fis-independent mutation appear to be completely dependent on Fis for this inversion. The corresponding mutations are located in different domains of the protein. The properties of these mutants in connection with the role of Fis in inversion will be discussed.