Mohinder K. Sardana

Regulation of JNK signaling by GSTp.

Studies of low basal Jun N‐terminal kinase (JNK) activity in non‐stressed cells led us to identify a JNK inhibitor that was purified and identified as glutathione S‐transferase Pi (GSTp) and was characterized as a JNK‐associated protein. UV irradiation or H2O2 treatment caused GSTp oligomerization and dissociation of the GSTp–JNK complex, indicating that it is the monomeric form of GSTp that elicits JNK inhibition. Addition of purified GSTp to the Jun–JNK complex caused a dose‐dependent inhibition of JNK activity. Conversely, immunodepleting GSTp from protein extracts attenuated JNK inhibition. Furthermore, JNK activity was increased in the presence of specific GSTp inhibitors and a GSTp‐derived peptide. Forced expression of GSTp decreased MKK4 and JNK phosphorylation which coincided with decreased JNK activity, increased c‐Jun ubiquitination and decreased c‐Jun‐mediated transcription. Co‐transfection of MEKK1 and GSTp restored MKK4 phosphorylation but did not affect GSTp inhibition of JNK activity, suggesting that the effect of GSTp on JNK is independent of the MEKK1–MKK4 module. Mouse embryo fibroblasts from GSTp‐null mice exhibited a high basal level of JNK activity that could be reduced by forced expression of GSTp cDNA. In demonstrating the relationships between GSTp expression and its association with JNK, our findings provide new insight into the regulation of stress kinases.

Photoactivated γ-secretase inhibitors directed to the active site covalently label presenilin 1

Cleavage of amyloid precursor protein (APP) by the β- and γ-secretases generates the amino and carboxy termini, respectively, of the Aβ amyloidogenic peptides Aβ40 and Aβ42—the major constituents of the amyloid plaques in the brain parenchyma of Alzheimers disease patients. There is evidence that the polytopic membrane-spanning proteins, presenilin 1 and 2 (PS1 and PS2), are important determinants of γ-secretase activity: mutations inxa0PS1 and PS2 that are associated with early-onset familial Alzheimers disease increase the production of Aβ42 (refs 4,5,6), the more amyloidogenic peptide; γ-secretase activity is reduced in neuronal cultures derived from PS1-deficient mouse embryos; and directed mutagenesis of two conserved aspartates in transmembrane segments of PS1 inactivates the ability of γ-secretase to catalyse processing of APP within its transmembrane domain. It is unknown, however, whether PS1 (which has little or no homology to any known aspartyl protease) is itself a transmembrane aspartyl protease or a γ-secretase cofactor, or helps to colocalize γ-secretase and APP. Here we report photoaffinity labelling of PS1 (and PS2) by potent γ-secretase inhibitors that were designed to function as transition state analogue inhibitors directed to the active site of an aspartyl protease. This observation indicates that PS1 (and PS2) may contain the active site of γ-secretase. Interestingly, the intact, single-chain form of wild-type PS1 is not labelled by an active-site-directed photoaffinity probe, suggesting that intact wild-type PS1 may be an aspartyl protease zymogen.

ATF2 confers radiation resistance to human melanoma cells.

We have previously identifed a U.V.-response element (URE; TGACAACA) and its bound proteins, members of the AP1 and ATF transcription factor families, in melanoma cells. Using a mutant form of cylic AMP response element binding (CREB), we found that CREB-associated-URE-bound proteins conferred characteristic melanoma phenotypes, including radiation resistance (Oncogene 12u2009:u20092223, 1996). In the present study we sought to determine which of the CREB-associated proteins confers radiation resistance on human melanoma cells. To this end we purified and identified via microsequencing ATF2 as a major URE- bound and CREB-associated protein in MeWo cells – a late stage human melanoma cell line. To determine the contribution of ATF2 to radiation resistance, MeWo cells were transfected with ATF2 cDNA lacking the trans-activation domain (ATF2Δ1u2009–u2009195). MeWo cells that stably express ATF2Δ1u2009–u2009195 showed weaker transcriptional activities and an altered pattern of homo/hetero dimers. ATF2Δ1-195 clones exhibited up to tenfold lower resistance to irradiation by either U.V. or X-rays. The degree of resistance to radiation in the ATF2Δ1u2009–u2009195-expressing clones could be increased upon transient transfection with ATF2wt, but not with phosphorylation-defective mutant ATF269,71. Similarly, transfection of ATF2wt to WM3211, an early stage human melanoma cells line, increased resistance to radiation. Finally, changes elicited through ATF2Δ1u2009–u2009195 also led to reduced drug resistance, as shown for MMC, araC and cis-platinum. Our results suggest that ATF2 is a regulator of radiation and drug resistance in melanomas, and that tumor targeted ATF2 modulators may be useful sensitizers in the treatment of tumors of this type.

Yeast thioltransferase--the active site cysteines display differential reactivity.

Thioltransferase, catalyzing thiol-disulfide interchange between reduced glutathione and disulfides, was purified to homogeneity from Saccharomyces cerevisiae. The purification procedure included ammonium sulfate precipitation, Sephadex G-50 gel filtration, CM-Sepharose ion exchange chromatography, and C18 reverse phase high pressure liquid chromatography. Two thioltransferase activity peaks were resolved by CM-Sepharose chromatography. The protein from the major peak had a molecular weight of 12 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis while the minor peak protein migrated slightly faster in this gel system. Both proteins showed similar amino acid compositions and identical N-termini. The major peak of thioltransferase was extensively characterized. Plots of thioltransferase activity as a function of S-sulfocysteine or hydroxyethyl disulfide concentration did not show normal Michaelis-Menten kinetics. The enzyme activity had a pH optimum of 9.1. The protein has 106 amino acid residues with two cysteines and no arginine. The active site amino acid sequence of the enzyme was identified as Cys26-Pro-Tyr-Cys29, which is similar to that of mammalian thioltransferase and Escherichia coli glutaredoxin. The two cysteines at the active site displayed different reactivities to iodoacetamide. Cys26 was alkylated by iodoacetamide at pH 3.5 while Cys29 was alkylated at pH 8.0. The enzyme was completely inactivated when the Cys26 was carboxymethylated. A plot of incorporation of iodoacetamide into Cys29 at different pHs was similar to the pH dependence of the enzyme activity. The result suggested that Cys26 could readily initiate nucleophilic attack on disulfide substrates at physiological pH.

Invertebrate aspartyl/asparaginyl β-hydroxylase: Potential modification of endogenous epidermal growth factor-like modules

An invertebrate alpha-ketoglutarate-dependent aspartyl/asparaginyl beta-hydroxylase, which posttranslationally hydroxylates specific aspartyl or asparaginyl residues within epidermal growth factor-like modules, was identified, partially purified and characterized. Preparations derived from two insect cell lines catalyzed the hydroxylation of the expected asparaginyl residue within a synthetic epidermal growth factor-like module. This activity was found to be similar to that of the purified mammalian aspartyl/asparaginyl beta-hydroxylase with respect to cofactor requirements, stereochemistry and substrate sequence specificity. Furthermore, recombinant human C1r, expressed in an insect cell-derived baculovirus expression system, was also found to be hydroxylated at the expected asparaginyl residue. Thus, these results establish the potential for invertebrate aspartyl/asparaginyl hydroxylation. Since several invertebrate proteins known to be required for proper embryonic development contain a putative consensus sequence that may be required for hydroxylation, the studies presented here provide the basis for further investigations concerned with identifying hydroxylated invertebrate proteins and determining their physiologic function.

Aldolase-DNA interactions in a SEWA cell system

In this report we demonstrate the novel finding that aldolase A interacts with DNA sequences in mouse SEWA sarcoma cells. This interaction was initially observed through the identification of a 40 kDa protein which was eluted from a DNA affinity chromatography column consisting of the long terminal repeat (LTR) of the endogenous intracisternal A-type particle (IAP). Microsequencing analysis identified this 40 kDa protein as the glycolytic enzyme, aldolase A. The use of specific anti-aldolase antibodies enabled the identification and subsequent purification of aldolase from the nuclear protein fraction of two SEWA sublines, one that is adherent and one that grows in suspension. In order to confirm our initial finding that aldolase is capable of interacting with DNA, proteins from each subline were immunopurified with anti-aldolase antibodies, eluted and then tested for their ability to interact with IAP-LTR DNA sequences. Interestingly, only aldolase derived from the anchorage dependent SEWA cells was capable of interacting with the IAP-LTR, however, several cell lines derived from human tumors also exhibited this activity. Subsequent studies revealed the ability of aldolase to interact with some but not every DNA sequence tested, implying that there may be a minimal DNA conformation and/or sequence requirement for this activity. The presence of aldolase A in the nuclei and its ability to interact with certain DNA sequences suggest a novel role for this metabolic enzyme.

Complete amino acid sequence of yeast thioltransferase (glutaredoxin)

The amino acid sequence of a thioltransferase isolated from Saccharomyces cerevisiae was determined. The protein was cleaved by trypsin, Staphylococcus aureus V8 protease, and cyanogen bromide. The peptides generated were purified by reverse phase HPLC. Sequencing of intact protein and its fragments were achieved by automated Edman degradation. The protein contains 106 amino acid residues with two cysteines. Yeast thioltransferase showed 51% structural similarity to pig liver thioltransferase and 34% to E. coli glutaredoxin.

Novel mutations introduced at the β-site of amyloid β protein precursor enhance the production of amyloid β peptide by BACE1 in vitro and in cells

Abnormal production and accumulation of amyloid-beta peptide (Abeta) plays a major role in the pathogenesis of Alzheimers disease (AD). beta-secretase (BACE1) is responsible for the cleavage at thebeta-site in amyloid beta protein precursor (AbetaPP/APP) to generate the N-terminus of Abeta. Here we report the stepwise identification and characterization of a novel APP-beta-site mutant, NFEV (APP_NFEV) in vitro and in cells. In vitro, the APP_NFEV exhibits 100-fold enhanced cleavage rate relative to the wild-type substrate (APPwt) and 10-fold increase relative to the Swedish-type mutation variant (APPsw). In cells, it was preferably cleaved among 24 APP beta-site mutations tested. More importantly, the APP_NFEV mutant failed to generate any detectable Abeta peptides in BACE1-KO mouse fibroblast cells. The production of Abeta peptides was restored by co-transfecting human BACE1, demonstrating that BACE1 is the only enzyme responsible for the processing of APP_NFEV in these cells. Analysis of APP_NFEV cleavage products secreted in the media revealed that in cells BACE1 cleaves APP_NFEV at the position between NF and EV, identical to that observed in vitro. A BACE inhibitor blocked the processing of the APP_NFEV beta-site in vitro and in cells. Our data indicates that the NFEV mutant is not only an enhanced substrate for BACE1 in vitro, but also a specific substrate for BACE1 in cells.

Design and Synthesis of a Selective PSA Cleavable Peptide-Doxorubicin Prodrug Which Targets PSA Positive Tumor Cells

Prostate cancer is the second leading cause of cancer mortality in males. It is estimated that 37,000 men died of prostate carcinoma in the United States in 1999 [1]. While cancer that is confined to the prostate can be treated with surgery or radiation, the prognosis for metastatic disease is poor.