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M R Pincus

Publications and source records attributed to M R Pincus.

At least 19 recordsLinked to original sources
PubMed2001

Peptides from the amino terminal mdm-2-binding domain of p53, designed from conformational analysis, are selectively cytotoxic to transformed cells.

We have synthesized three peptides from the mdm-2 binding domain of human p53, residues 12-26 (PPLSQETFSDLWKLL), residues 12-20, and 17-26. To enable transport of the peptides across the cell membrane and at the same time to maximize the active mdm-2 binding alpha-helical conformation for these peptides, each was attached at its carboxyl terminus to the penetratin sequence, KKWKMRRNQFWVKVQRG, that contains many positively charged residues that stabilize an alpha-helix when present on its carboxyl terminal end. All three peptides were cytotoxic to human cancer cells in culture, whereas a control, unrelated peptide attached to the same penetratin sequence had no effect on these cell lines. The same three cytotoxic peptides had no effect on the growth of normal cells, including human cord blood-derived stem cells. These peptides were as effective in causing cell death in p53-null cancer cells as in those having mutant or normal p53. Peptide-induced cell death is not accompanied by expression of apoptosis-associated proteins such as Bax and waf(p21). Based on these findings, we conclude that the antiproliferative effects of these p53-derived peptides are not completely dependent on p53 activity and may prove useful as general anticancer agents.

Amino Acid Sequence↗
PubMed2001

Differences in patterns of activation of MAP kinases induced by oncogenic ras-p21 and insulin in oocytes.

Oncogenic ras (Val 12-containing)-p21 protein induces oocyte maturation by a pathway that is blocked by peptides from effector domains of ras-p21, i.e., residues 35-47 (that block Val 12-p21-activated raf) and 96-110 and 115-126, which do not affect the ability of insulin-activated cellular p21 to induce maturation. Oncogenic p21 binds directly to jun-N-terminal kinase (JNK), which is blocked by the p21 96-110 and 115-126 peptides. This finding predicts that oncogenic p21, but not insulin, induces maturation by early and sustained activation of JNK. We now directly confirm this prediction by showing that oncogenic p21 induces activating phosphorylation of JNK (JNK-P) and of ERK (MAP kinase) (MAPK-P), whose levels correlate with oocyte maturation. p21 peptides 35-47 and 96-110 block formation of JNK-P and MAPK-P, further confirming this correlation and suggesting, unexpectedly, that raf-MEK-MAPK and JNK-jun pathways strongly interact on the oncogenic p21 pathway. In contrast, insulin activates only low levels of JNK-P, and, surprisingly, we find that insulin induces only low levels of MAPK-P, indicating that insulin and activated normal p21 utilize MAP kinase-independent signal transduction pathways.

Animals↗
PubMed2001

Plasmid expression of a peptide that selectively blocks oncogenic ras-p21-induced oocyte maturation.

PURPOSE: We have previously found that a synthetic peptide corresponding to ras-p21 residues 96 110 (PNC2) selectively blocks oncogenic (Val 12-containing) ras-p21 protein-induced oocyte maturation. With a view to introducing this peptide into ras-transformed human cells to inhibit their proliferation, we synthesized an inducible plasmid that expressed this peptide sequence. Our purpose was to test this expression system in oocytes to determine if it was capable of causing selective inhibition of oncogenic ras-p21. METHODS: We injected this plasmid and a plasmid expressing a control peptide into oocytes either together with oncogenic p21 or in the presence of insulin (that induces maturation that is dependent on normal cellular ras-p21) in the presence and absence of the inducer isopropylthioglucose (IPTG). RESULTS: Microinjection of this plasmid into oocytes together with Val 12-p21 resulted in complete inhibition of maturation in the presence of inducer. Another plasmid encoding the sequence for the unrelated control peptide, X13, was unable to inhibit Val 12-p21-induced maturation. In contrast, PNC2 plasmid had no effect on the ability of insulin-activated normal cellular or wild-type ras-p21 to induce oocyte maturation, suggesting that it is selective for blocking the mitogenic effects of oncogenic (Val 12) ras p21. CONCLUSION: We conclude that the PNC2 plasmid selectively inhibits oncogenic ras-p21 and may therefore be highly effective in blocking proliferation of ras-induced cancer cells. Also, from the patterns of inhibition, by PNC2 and other ras- and raf-related peptides, of raf- and constitutively activated MEK-induced maturation, we conclude that PNC2 peptide inhibits oncogenic ras p21 downstream of raf.

Amino Acid Sequence↗
PubMed2001

Common conformational effects of p53 mutations.

The tumor suppressor gene p53 has been identified as the most frequent target of genetic alterations in human cancers. Most of these mutations occur in highly conserved regions in the DNA-binding core domain of the p53 protein, suggesting that the amino acid residues in these regions are critical for maintaining normal p53 structure and function. We previously used molecular dynamics calculations to demonstrate that several amino acid substitutions in these regions that are induced by environmental carcinogens and found in human tumors produce certain common conformational changes in the mutant proteins that differ substantially from the wild-type structure. In order to determine whether these conformational changes are consistent for other p53 mutants, we have now used molecular dynamics to determine the structure of the DNA-binding core domain of seven other environmentally induced, cancer-related p53 mutants, namely His 175, Asp 245, Asn 245, Trp 248, Met 249, Ser 278, and Lys 286. The results indicate that all of these mutants differ substantially from the wild-type structure in certain discrete regions and that some of these conformational changes are similar for these mutants as well as those determined previously. The changes are also consistent with experimental evidence for alterations in structure in p53 mutants determined by epitope detectability using monoclonal antibodies directed against these regions of predicted conformational change.

Amino Acid Substitution↗
PubMed2001

Jun NH2-terminal kinase phosphorylation of p53 on Thr-81 is important for p53 stabilization and transcriptional activities in response to stress.

The p53 tumor suppressor protein plays a key role in the regulation of stress-mediated growth arrest and apoptosis. Stress-induced phosphorylation of p53 tightly regulates its stability and transcriptional activities. Mass spectrometry analysis of p53 phosphorylated in 293T cells by active Jun NH2-terminal kinase (JNK) identified T81 as the JNK phosphorylation site. JNK phosphorylated p53 at T81 in response to DNA damage and stress-inducing agents, as determined by phospho-specific antibodies to T81. Unlike wild-type p53, in response to JNK stimuli p53 mutated on T81 (T81A) did not exhibit increased expression or concomitant activation of transcriptional activity, growth inhibition, and apoptosis. Forced expression of MKP5, a JNK phosphatase, in JNK kinase-expressing cells decreased T81 phosphorylation while reducing p53 transcriptional activity and p53-mediated apoptosis. Similarly transfection of antisense JNK 1 and -2 decreased T81 phosphorylation in response to UV irradiation. More than 180 human tumors have been reported to contain p53 with mutations within the region that encompasses T81 and the JNK binding site (amino acids 81 to 116). Our studies identify an additional mechanism for the regulation of p53 stability and functional activities in response to stress.

Animals↗
PubMed2000

Molecular modeling of mammalian cytochrome P450s.

The cytochrome P450s are a superfamily of hemoprotein enzymes responsible for the metabolism of a wide variety of xenobiotic and endogenous compounds. The individual P450s exhibit unique substrate specificity and stereoselectivity profiles which reflect corresponding differences in primary sequence and tertiary structure. In the absence of an experimental structure models for mammalian P450s have been generated by their homology with bacterial P450s of known structure. The rather low sequence identity between target and template proteins renders P450 modeling a challenging task. However, the substrate recognition properties of several P450s are consistent with recently developed working models. This review summarizes the major concepts and current approaches of molecular modeling of P450s.

Amino Acid Sequence↗
PubMed2000

Induction of oocyte maturation by jun-N-terminal kinase (JNK) on the oncogenic ras-p21 pathway is dependent on the raf-MEK signal transduction pathway.

PURPOSE: We have previously found that microinjection of activated MEK (mitogen activated kinase kinase) and ERK (mitogen-activated protein; MAP kinase) fails to induce oocyte maturation, but that maturation, induced by oncogenic ras-p21 and insulin-activated cell ras-p21, is blocked by peptides from the ras-binding domain of raf. We also found that jun kinase (JNK), on the stress-activated protein (SAP) pathway, which is critical to the oncogenic ras-p21 signal transduction pathway, is a strong inducer of oocyte maturation. Our purpose in this study was to determine the role of the raf-MEK-MAP kinase pathway in oocyte maturation and how it interacts with JNK from the SAP pathway. METHODS: We microinjected raf dominant negative mutant mRNA (DN-raf) and the MEK-specific phosphatase, MKP-T4, either together with oncogenic p21 or c-raf mRNA, into oocytes or into oocytes incubated with insulin to determine the effects of these raf-MEK-MAP kinase pathway inhibitors. RESULTS: We found that oocyte maturation induced by both oncogenic and activated normal p21 is inhibited by both DN-raf and by MKP-T4. The latter more strongly blocks the oncogenic pathway. Also an mRNA encoding a constitutively activated MEK strongly induces oocyte maturation that is not inhibited by DN-raf or by MKP-T4. Surprisingly, we found that oocyte maturation induced by JNK is blocked both by DN-raf and MKP-T4. Furthermore, we discovered that c-raf induces oocyte maturation that is inhibited by glutathione-S-transferase (GST), which we have found to be a potent and selective inhibitor of JNK. CONCLUSION: We conclude that there is a strong reciprocal interaction between the SAP pathway involving JNK and the raf-MEK-MAP kinase pathway and that oncogenic ras-p21 can be preferentially inhibited by MEK inhibitors. The results imply that blockade of both MEK and JNK-oncogenic ras-p21 interactions may constitute selective synergistic combination chemotherapy against oncogenic ras-induced tumors.

Animals↗
PubMed2000

Identification, using molecular dynamics, of an effector domain of the ras-binding domain of the raf-p74 protein that is uniquely involved in oncogenic ras-p21 signaling.

By comparing the average structures, computed using molecular dynamics, of the ras-binding domain of raf (RBD) bound to activated wild-type ras-p21 and its homologous inhibitory protein, rap-1A, we formerly identified three domains of the RBD that changed conformation between the two complexes, residues 62-76, 97-110, and 111-121. We found that one synthetic peptide, corresponding to RBD residues 97-110, selectively inhibited oncogenic ras-p21-induced oocyte maturation. In this study, we performed molecular dynamics on the Val 12-ras-p21-RBD complex and compared its average structure with that for the wild-type protein. We find that there is a large displacement of a loop involving these residues when the structures of the two complexes are compared. This result corroborates our former finding that the RBD 97-110 peptide inhibits only signal transduction by oncogenic ras-p21 and suggests that oncogenic p21 uses this loop to interact with raf in a unique manner.

Amino Acid Substitution↗
PubMed2000

Glutathione-S-Transferase as a selective inhibitor of oncogenic ras-p21-induced mitogenic signaling through blockade of activation of jun by jun-N-terminal kinase.

We have identified the intracellular detoxification enzyme, glutathione-S-transferase (GST), as a potent inhibitor of the activation of jun by its kinase, jun-N-terminal kinase (JNK), in vitro. All three major isozymes (alpha, mu, and pi) bind to JNK-jun complexes and inhibit activation of jun by JNK. We now find that GST inhibits JNK-induced oocyte maturation in vivo and strongly inhibits oocyte maturation induced by oncogenic ras-p21 protein, but not by insulin-activated normal cellular p21 protein. These results correlate with the finding that oncogenic, but not insulin-activated normal, p21 induces high levels of activated JNK. GST also strongly blocks induction of oocyte maturation by protein kinase C (PKC) which is a critical downstream target of oncogenic but not normal ras-p21. Thus, we have established a new function for GST as a potent physiological inhibitor of the ras-JNK-jun pathway.

Animals↗
PubMed1999

Conformational and molecular basis for induction of apoptosis by a p53 C-terminal peptide in human cancer cells.

A p53-derived C-terminal peptide induced rapid apoptosis in breast cancer cell lines carrying endogenous p53 mutations or overexpressed wild-type (wt) p53 but was not toxic to nonmalignant human cell lines containing wt p53. Apoptosis occurred through a Fas/APO-1 signaling pathway involving increased extracellular levels of Fas/FasL in the absence of protein synthesis, as well as activation of a Fas/APO-1-specific protease, FLICE. The peptide activity was p53-dependent, and it had no effect in three tumor cell lines with null p53. Furthermore, the C-terminal peptide bound to p53 protein in cell extracts. Thus, p53-dependent, Fas/APO-1 mediated apoptosis can be induced in breast cancer cells with mutant p53 similar to the recently described Fas/APO-1 induced apoptosis by wt p53. However, mutant p53 without p53 peptide does not induce a Fas/APO-1 activation or apoptosis. Docking of the computed low energy conformations for the C-terminal peptide with those for a recently defined proline-rich regulatory region from the N-terminal domain of p53 suggests a unique low energy complex between the two peptide domains. The selective and rapid induction of apoptosis in cancer cells carrying p53 abnormalities may lead to a novel therapeutic modality.

Amino Acid Sequence↗
PubMed1999

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.

3T3 Cells↗
PubMed1999

Molecular dynamics analysis of the structures of ras-guanine nucleotide exchange protein (SOS) bound to wild-type and oncogenic ras-p21. Identification of effector domains of SOS.

The X-ray crystal structure of the ras oncogene-encoded p21 protein bound to SOS, the guanine nucleotide exchange-promoting protein, has been determined. We have undertaken to determine if there are differences between the three-dimensional structures of SOS bound to normal and oncogenic (Val 12-p21) proteins. Using molecular dynamics, we have computed the average structures for both complexes and superimposed them. We find four domains of SOS that differ markedly in structure: 631-641, 676-691, 718-729, and 994-1004. Peptides corresponding to these sequences have been synthesized and found to be powerful modulators of oncogenic p21 in cells as described in an accompanying paper. We find that the SOS segment from 809-815 makes contacts with multiple domains of ras-p21 and can facilitate correlated conformational changes in these domains.

Models, Molecular↗
PubMed1999

Identification of the site of inhibition of oncogenic ras-p21-induced signal transduction by a peptide from a ras effector domain.

We have previously found that a peptide corresponding to residues 35-47 of the ras-p21 protein, from its switch 1 effector domain region, strongly inhibits oocyte maturation induced by oncogenic p21, but not by insulin-activated cellular wild-type p21. Another ras-p21 peptide corresponding to residues 96-110 that blocks ras-jun and jun kinase (JNK) interactions exhibits a similar pattern of inhibition. We have also found that c-raf strongly induces oocyte maturation and that dominant negative c-raf strongly blocks oncogenic p21-induced oocyte maturation. We now find that the p21 35-47, but not the 96-110, peptide completely blocks c-raf-induced maturation. This finding suggests that the 35-47 peptide blocks oncogenic ras at the level of raf; that activated normal and oncogenic ras-p21 have differing requirements for raf-dependent signaling; and that the two oncogenic-ras-selective inhibitory peptides, 35-47 and 96-110, act at two different critical downstream sites, the former at raf the latter at JNK/jun, both of which are required for oncogenic ras-p21 signaling.

Animals↗
PubMed1999

Common conformational effects in the p53 protein of vinyl chloride-induced mutations.

The tumor suppressor gene p53 has been identified as the most frequent site of genetic alterations in human cancers. Vinyl chloride, a known human carcinogen, has been associated with specific A --> T transversions at codons 179, 249, and 255 of the p53 gene. The mutations result in amino acid substitutions of His --> Leu at residue 179, Arg --> Trp at residue 249, and He --> Phe at residue 255 in highly conserved regions of the DNA-binding core domain of the p53 protein. We previously used molecular dynamics calculations to demonstrate that the latter two mutants contain certain common regions that differ substantially in conformation from the wild-type structure. In order to determine whether these conformational changes are consistent for other p53 mutants, we have now used molecular dynamics to determine the structure of the DNA-binding core domain of the Leu 179 p53 mutant. The results indicate that the Leu 179 mutant differs substantially from the wild-type structure in certain discrete regions that are similar to those noted previously in the other p53 mutants. One of these regions (residues 204-217) contains the epitope for the monoclonal antibody PAb240, which is concealed in the wild-type structure, but accessible in the mutant structure, and another region (residues 94-110) contains the epitope for the monoclonal antibody PAb1620, which is accessible in the wild-type structure, but concealed in the mutant structure. Immunologic analyses of tumor tissue known to contain this mutation confirmed these predicted conformational shifts in the mutant p53 protein.

Animals↗
PubMed1999

Identification of a glutathione-S-transferase effector domain for inhibition of jun kinase, by molecular dynamics.

We have recently found that the glutathione-S-transferase pi-isozyme (GST-pi), a cellular detoxification enzyme, potently and selectively inhibits activation of jun protein by its upstream kinase, jun kinase (JNK). This newly identified regulatory activity of GST-pi is strongly inhibited by a group of agents that inhibit its enzymatic activity. Since loss of enzymatic activity in general does not correlate with loss of regulatory activity, it is likely that inhibitor binding induces changes in the structure of one or more domains of GST that block its interaction with JNK. To identify regions of GST that change conformation on the binding of inhibitors, we have performed molecular dynamics calculations on GST-pi to compute its average structure in the presence and absence of the inhibitor, glutathione sulfonate. Superposition of the two average structures reveals that several regions change local structure depending upon whether the inhibitor is bound or not bound. Two of these regions, residues 36-50 and 194-201, are highly exposed. We have synthesized peptides corresponding to these two segments and find that the 194-201 sequence strongly inhibits the ability of GST-pi to block the in vitro phosphorylation of jun by JNK. These results suggest that this region of GST-pi is critical to its functioning as a newly discovered regulator of signal transduction.

Amino Acid Sequence↗
PubMed1999

Inhibition of oncogenic and activated wild-type ras-p21 protein-induced oocyte maturation by peptides from the guanine-nucleotide exchange protein, SOS, identified from molecular dynamics calculations. Selective inhibition of oncogenic ras-p21.

In the preceding paper we performed molecular dynamics calculations of the average structures of the SOS protein bound to wild-type and oncogenic ras-p21. Based on these calculations, we have identified four major domains of the SOS protein, consisting of residues 631-641, 676-691, 718-729, and 994-1004, which differ in structure between the two complexes. We have now microinjected synthetic peptides corresponding to each of these domains into Xenopus laevis oocytes either together with oncogenic (Val 12)-p21 or into oocytes subsequently incubated with insulin. We find that the first three peptides inhibit both oncogenic and wild-type p21-induced oocyte maturation, while the last peptide much more strongly inhibits oncogenic p21 protein-induced oocyte maturation. These results suggest that each identified SOS region is involved in ras-stimulated signal transduction and that the 994-1004 domain is involved uniquely with oncogenic ras-p21 signaling.

Amino Acid Sequence↗
PubMed1998

MEKK1/JNK signaling stabilizes and activates p53.

Activation of the tumor suppressor p53 by stress and damage stimuli often correlates with induction of stress kinases, Jun-NH2 kinase (JNK). As JNK association with p53 plays an important role in p53 stability, in the present study we have elucidated the relationship between the JNK-signaling pathway and p53 stability and activity. Expression of a constitutively active form of JNKK upstream kinase, mitogen-activated protein kinase kinase kinase (DeltaMEKK1), increased the level of the exogenously transfected form of p53 in p53 null (10.1) cells as well as of endogenous p53 in MCF7 breast cancer cells. Increased p53 level by forced expression of DeltaMEKK1 coincided with a decrease in p53 ubiquitination in vivo and with prolonged p53 half-life. Computerized modeling of the JNK-binding site (amino acids 97-116; p7 region) enabled us to design mutations of exposed residues within this region. Respective mutations (p53(101-5-8)) and deletion (p53(Deltap7)) forms of p53 did not exhibit the same increase in p53 levels upon DeltaMEKK1 expression. In vitro phosphorylation of p53 by JNK abolished Mdm2 binding and targeting of p53 ubiquitination. Similarly, DeltaMEKK1 expression increased p53 phosphorylation by immunopurified JNK and dissociated p53-Mdm2 complexes. Transcriptional activity of p53, as measured via mdm2 promoter-driven luciferase, exhibited a substantial increase in DeltaMEKK1-expressing cells. Cotransfection of p53 and DeltaMEKK1 into p53 null cells potentiated p53-dependent apoptosis, suggesting that MEKK1 effectors contribute to the ability of p53 to mediate programmed cell death. Our results point to the role of MEKK1-JNK signaling in p53 stability, transcriptional activities, and apoptotic capacity as part of the cellular response to stress.

Apoptosis↗