Human immunodeficiency virus (HIV) antigens and RNA in HIV-seronegative women with cervical intraepithelial neoplasia.

While investigating whether proteins retrieved by cervicovaginal lavages (CVL) from women with cervical intraepithelial neoplasia (CIN) might correlate with risk of progression to invasive cervical cancer, we unexpectedly identified HIV gag and env glycoprotein in CVL from women with HIV-negative serology. HIV antigens were consistently identified by mass spectrometry (MS) in CVL from 4 women but were absent in CVL from the remaining 16 women. HIV serologies of all 20 patients were negative for both HIV-1 and HIV-2 antibodies. To validate the unexpected MS findings we performed Western blot (WB) and immunoaffinity chromatography (IC) analysis of CVL for HIV proteins, viral load assays of paired CVL and blood samples, and immunohistochemical HIV p24 expression in cervical biopsy specimens. WB analysis of CVL for prostate-specific antigen (PSA) was performed to exclude semen contamination as the source of HIV proteins. WB and IC results demonstrated the presence of HIV-1 gp41 and p24 antigens in four CVL that were identified by MS to have the HIV proteins. Despite negative serology, HIV RNA in CVL and HIV p24 in cervix biopsies were detected in patients with HIV antigen-positive CVL. HIV p24-positive CVL were PSA negative. All 20 subjects remained HIV seronegative throughout the study. Women with HIV proteins and RNA were comparatively older. Our findings suggest that CVL HIV proteins in women with CIN could be markers for unrecognized HIV exposure or subclinical infection. Proteomic screening of cervical secretions may be useful in identifying seronegative women exposed to HIV and/or at risk for AIDS.

A photoaffinity analogue of discodermolide specifically labels a peptide in beta-tubulin.

Discodermolide is a potentially important antitumor agent that stabilizes microtubules and blocks cells at the G2/M phase of the cell cycle in a manner similar to that of Taxol. Discodermolide also has unique properties that distinguish it from Taxol. In the present study, photoaffinity-labeled discodermolide analogues are used to investigate their binding site in tubulin. Three photoaffinity-labeled discodermolide analogues were synthesized, all of which promoted microtubule polymerization in the absence of GTP. The analogue, C19-[4-(4-(3)H-benzoyl-phenyl)-carbamate]-discodermolide (C19-[3H]BPC-discodermolide), was selected for photolabeling studies because it had the highest extent of photoincorporation, approximately 1%, of the three radiolabeled discodermolide analogues explored. Although compared to discodermolide, C19-BPC-discodermolide revealed no hypernucleation effect in the in vitro microtubule polymerization assay, it was more cytotoxic than discodermolide, and, like discodermolide, demonstrated synergism with Taxol. These results suggest that the hypernucleation effect of discodermolide is not involved in its cytotoxic activity. Similar to discodermolide, C19-BPC-discodermolide can effectively displace [3H]Taxol from microtubules, but Taxol cannot effectively displace C19-[3H]BPC-discodermolide binding. Discodermolide can effectively displace C19-[3H]BPC-discodermolide binding. Formic acid hydrolysis, immunoprecipitation experiments, and subtilisin digestion indicate that C19-BPC-discodermolide labels amino acid residues 305-433 in beta-tubulin. Further digestion with Asp-N and Arg-C enzymes suggested that C19-BPC-discodermolide binds to amino acid residues, 355-359, in beta-tubulin, which is in close proximity to the Taxol binding site. Molecular modeling guided by the above evidence led to a putative binding model for C19-BPC-discodermolide in tubulin.

Insights into the mechanism of microtubule stabilization by Taxol.

The antitumor drug Taxol stabilizes microtubules and reduces their dynamicity, promoting mitotic arrest and cell death. Upon assembly of the alpha/beta-tubulin heterodimer, GTP bound to beta-tubulin is hydrolyzed to GDP reaching a steady-state equilibrium between free tubulin dimers and microtubules. The binding of Taxol to beta-tubulin in the polymer results in cold-stable microtubules at the expense of tubulin dimers, even in the absence of exogenous GTP. However, there is little biochemical insight into the mechanism(s) by which Taxol stabilizes microtubules. Here, we analyze the structural changes occurring in both beta- and alpha-tubulin upon microtubule stabilization by Taxol. Hydrogen/deuterium exchange (HDX) coupled to liquid chromatography-electrospray ionization MS demonstrated a marked reduction in deuterium incorporation in both beta-and alpha-tubulin when Taxol was present. Decreased local HDX in peptic peptides was mapped on the tubulin structure and revealed both expected and new dimer-dimer interactions. The increased rigidity in Taxol microtubules was distinct from and complementary to that due to GTP-induced polymerization. The Taxol-induced changes in tubulin conformation act against microtubule depolymerization in a precise directional way. These results demonstrate that HDX coupled to liquid chromatography-electrospray ionization MS can be effectively used to study conformational effects induced by small ligands on microtubules. The present study also opens avenues for locating drug and protein binding sites and for deciphering the mechanisms by which their interactions alter the conformation of microtubules and tubulin dimers.

Rat organic anion transporting protein 1A1 (Oatp1a1): purification and phosphopeptide assignment.

Rat organic anion transporting protein 1a1 (oatp1a1), a hepatocyte basolateral plasma membrane protein, mediates transport of various amphipathic compounds. Our previous studies indicated that serine phosphorylation of a single tryptic peptide inhibits its transport activity without changing its cell surface content. The site of phosphorylation is unknown and was the subject of the present study. Following immunoaffinity chromatographic purification from rat liver, oatp1a1 was subjected to trypsin digestion and MALDI-TOF. Except for predicted N-glycosylated peptides, 97% of oatp1a1 tryptic peptides were observed. A single tryptic phosphopeptide was found in the C-terminus (aa 626-647), existing in unphosphorylated or singly or doubly phosphorylated forms and sensitive to alkaline phosphatase treatment. The beta-elimination reaction resulted in a mass loss of 98 or 196 Da from this peptide, and subsequent Michael addition with cysteamine increased masses by the predicated 77 and 154 Da, indicating that oatp1a1 can be singly or doubly phosphorylated at serine or threonine residues in the C-terminal sequence SSATDHT (aa 634-640). Subsequent tandem MS/MS analysis revealed that phosphorylation at S634 accounted for all singly phosphorylated peptide, while phosphorylation at S634 and S635 accounted for all doubly phosphorylated peptide. These findings identify the site of oatp1a1 phosphorylation and demonstrate that it is an ordered process, in which phosphorylation at S634 precedes that at S635. The mechanism by which phosphorylation results in loss of transport activity in hepatocytes remains to be established. Whether phosphorylation near the C-terminus inhibits C-terminal oligomerization of oatp1a1, required for normal transport function, can be speculated upon but is as yet unknown.

Detection of human betaV-tubulin expression in epithelial cancer cell lines by tubulin proteomics.

Tubulin, the constitutive protein of microtubules, is a heterodimeric protein with an alpha and beta subunit, encoded in vertebrates by six and seven different genes, respectively. Each tubulin isotype can be identified by its divergent C-terminal sequence. Nevertheless, two groups of beta-tubulin isotypes can be distinguished by sequence alignment; one includes betaI-, betaII-, betaIVa-, and betaIVb-tubulin, and the other includes betaIII-, betaV-, and betaVI-tubulin. betaIII-tubulin overexpression has been associated with microtubule destabilization and resistance to Taxol. Recent data indicate that mouse betaV-tubulin overexpression in CHO cells results in profound microtubule disorganization and dependence of cells on Taxol for growth. Mouse and human betaV-tubulin sequences display several differences, such as their respective extreme C-terminus, suggesting that they may have different effects on microtubule stability and different affinities for drugs. When high-resolution isoelectric focusing, in-gel CNBr cleavage, and mass spectrometry were combined, we detected for the first time the betaV-tubulin protein in human cell lines and found that it was highly expressed in Hey, an epithelial ovarian cancer cell line. Our data confirm that human and rodent betaV-tubulins are distinct and indicate that, regardless of species, betaIII- and betaV-tubulin may be expressed in a complementary pattern at the protein level. Therefore, both betaIII- and betaV-tubulin expression levels should be systematically determined to assess the role of differential tubulin isotype expression in the response of tumors to drugs targeting microtubules.

Interaction with PDZK1 is required for expression of organic anion transporting protein 1A1 on the hepatocyte surface.

Although many organic anion transport protein (Oatp) family members have PDZ consensus binding sites at their C termini, the functional significance is unknown. In the present study, we utilized rat Oatp1a1 (NM_017111) as a prototypical member of this family to examine the mechanism governing its subcellular trafficking. A peptide corresponding to the C-terminal 16 amino acids of rat Oatp1a1 was used to affinity-isolate interacting proteins from rat liver cytosol. Protein mass fingerprinting identified PDZK1 as the major interacting protein. This was confirmed by immunoprecipitation of an Oatp1a1-PDZK1 complex from cotransfected 293T cells as well as from native rat liver membrane extracts. Oatp1a1 bound predominantly to the first and third PDZ binding domains of PDZK1, whereas the high density lipoprotein receptor, scavenger receptor B type I binds to the first domain. Although it is possible that PDZK1 forms a complex with these two integral membrane proteins, this did not occur, suggesting that as yet undescribed factors lead to selectivity in the interaction of these protein ligands with PDZK1. Oatp1a1 protein expression was near normal in PDZK1 knock-out mouse liver. However, it was located predominantly in intracellular structures, in contrast to its normal basolateral plasma membrane distribution. Plasma disappearance of the Oatp1a1 ligand [35S]sulfobromophthalein was correspondingly delayed in knock-out mice. These studies show a critical role for oligomerization of Oatp1a1 with PDZK1 for its proper subcellular localization and function. Because its ability to transport substances into the cell requires surface expression, this must be considered in any assessment of physiologic function.

A highly epothilone B-resistant A549 cell line with mutations in tubulin that confer drug dependence.

A 95-fold epothilone B (EpoB)-resistant, but not dependent, A549 human lung carcinoma cell line, A549.EpoB40 (EpoB40), has a Gln to Glu mutation at residue 292 that is situated near the M-loop of betaI-tubulin. Further selection of this cell line with higher concentrations of EpoB produced A549.EpoB480 (EpoB480), which is approximately 900-fold resistant to EpoB. This cell line, like EpoB40, exhibits cross-resistance to Taxol and extreme sensitivity to vinblastine, but in contrast to EpoB40 it is unusually dependent on EpoB, requiring a minimum of 125 nmol/L EpoB to maintain normal growth. Sequence analysis of the beta-tubulin and Kalpha1-tubulin genes in EpoB480 showed that, in addition to the beta292 mutation, beta60 was mutated from Val to Phe and alpha195 was mutated from Leu to Met. Mass spectrometry indicated that both the Val(60)Phe and Leu(195)Met mutations in betaI- and Kalpha1-tubulin, respectively, were expressed at the protein level. Molecular modeling indicated that beta60 is located at the end of the H1-S2 loop that has been implicated as a principal partner of the M-loop for contacts between protofilaments. A mutation at beta60 could inhibit the lateral contacts between protofilaments, thereby destabilizing microtubules. alpha195 is located at the external surface of the microtubule that has been proposed as the domain that interacts with a variety of endogenous proteins, such as stathmin and microtubule-associated protein 4. A mutation at alpha195 could modulate the interactions between tubulin and regulatory proteins. We propose that the betaVal(60)Phe mutation plays a critical role in the drug-dependent phenotype of EpoB480 cells.

Direct analysis of tubulin expression in cancer cell lines by electrospray ionization mass spectrometry.

Differential expression of tubulin isotypes, mutations, and/or post-translational modifications in sensitive and Taxol-resistant cell lines suggests the existence of tubulin-based mechanisms of resistance. Since tubulin isotypes are defined by their C-terminal sequence, we previously described a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based analysis of tubulin diversity in human cell lines by analysis of their CNBr-released C-terminal peptides [Rao, S., Aberg, F., Nieves, E., Horwitz, S. B., and Orr, G. A. (2001) Biochemistry 40, 2096-103]. We now describe the liquid chromatography/electrospray ionization mass spectrometry analysis of native tubulins in Taxol-stabilized microtubules from parental and Taxol/epothilone-resistant human cancer cell lines. This method allows the direct determination of tubulin isotype composition, including post-translational modifications and mutations occurring throughout the entire protein. Four major isotypes, betaI-, betaIVb-, Kalpha1-, and alpha6-tubulin, were detected in two human carcinoma cell lines, A549 and HeLa. betaIII-Tubulin represented a minor species, as did alpha4-tubulin which was detected for the first time in both cell lines. The three alpha-tubulins were almost totally tyrosinated, and post-translational modifications were limited to low levels of monoglutamylation of Kalpha1-, betaI-, and betaIII-tubulin. betaII- and betaIVa-tubulins were not detected in either parental or drug-resistant cell lines, in contrast to previous RNA-based studies. Since mutations can occur in a single tubulin allele, the question as to whether the wild-type and mutant transcripts are both translated, and to what levels, is important. Heterozygous expression of Kalpha1- or betaI-tubulin mutants that introduced mass changes as small as 26 Da was readily detected in native tubulins isolated from Taxol- and epothilone-resistant cell lines.

Mechanisms of Taxol resistance related to microtubules.

Since its approval by the FDA in 1992 for the treatment of ovarian cancer, the use of Taxol has dramatically increased. Although treatment with Taxol has led to improvement in the duration and quality of life for some cancer patients, the majority eventually develop progressive disease after initially responding to Taxol treatment. Drug resistance represents a major obstacle to improving the overall response and survival of cancer patients. This review focuses on mechanisms of Taxol resistance that occur directly at the microtubule, such as mutations, tubulin isotype selection and post-translational modifications, and also at the level of regulatory proteins. A review of tubulin structure, microtubule dynamics, the mechanism of action of Taxol and its binding site on the microtubule are included, so that the reader can evaluate Taxol resistance in context.

Analysis of tubulin isotypes and mutations from taxol-resistant cells by combined isoelectrofocusing and mass spectrometry.

Six human alpha-tubulin and seven human beta-tubulin isotypes, each of which can undergo posttranslational modifications, have been detected by the reverse transcriptase-polymerase chain reaction. This repertoire of tubulin isotypes plays a role in development and in the building of specialized microtubule-based structures. In cell lines, the relationship between resistance to microtubule-interacting drugs and altered tubulin isotype expression profiles is often established by quantitation of cDNA and/or Western blot analysis. Tubulin mutations in major isotypes are detected by sequencing cDNA, but more analysis of expression of tubulin mutations at the protein level, to assess their role in drug resistance, is needed. We utilized a Taxol-based purification and high-resolution isoelectrofocusing combined with a mass spectrometry-based analysis of tubulin. This approach has allowed the separation and relative quantitation of tubulin isotypes having a difference in isoelectric point values of 0.01, without the need for two-dimensional gel electrophoresis. The specificity of tubulin isotype antibodies also has been established. In cell lines resistant to microtubule-stabilizing drugs that express heterozygous tubulin mutations, the relative amount of mutant tubulin expression has been determined. In these cell lines, the absence of betaII- and betaIVa-tubulin has been demonstrated, and an increased level of expression of betaIII-tubulin in resistant cells has been confirmed, indicating that this tubulin isotype is a unique marker of resistance.

Elevated levels of microtubule destabilizing factors in a Taxol-resistant/dependent A549 cell line with an alpha-tubulin mutation.

The A549 Taxol-resistant cell lines, A549-T12 and A549-T24, were isolated in our laboratory, and are dependent on Taxol for normal growth. The microtubules in these cells displayed increased dynamicity in the absence of Taxol. In the present study, a heterozygous point mutation in Kalpha1-tubulin was discovered at alpha379 (Ser to Ser/Arg). Although Taxol binds to beta-tubulin in the microtubule, sequencing of beta-tubulin class I did not reveal any mutations. The expression of the alpha-tubulin mutation was demonstrated using high-resolution isoelectric focusing and two-dimensional gel analysis. Both the wild-type and mutant tubulin were expressed in the Taxol-resistant cell lines. The region of alpha-tubulin that encompasses alpha379 is near the COOH terminus that has been proposed as a site of interaction with microtubule-associated protein (MAP) 4 and stathmin, a tubulin-interacting protein. In the Taxol-resistant cells, the active nonphosphorylated form of stathmin was increased approximately 2-fold, whereas the inactive phosphorylated forms were barely detected. The inactive phosphorylated forms of MAP4 were increased in the A549-T12 and A549-T24 cell lines. We hypothesize that these changes in tubulin/MAPs that result in increased microtubule instability may be related to the alpha-tubulin mutation and are compensated for by the stabilizing properties of Taxol.

Molecular cloning and expression of the catalytic subunit of protein kinase A from Trypanosoma cruzi.

The activation of protein kinase A (cyclic adenosine monophosphate-dependent protein kinase) by cyclic adenosine monophosphate is believed to play an important role in regulating the growth and differentiation of Trypanosoma cruzi. A PCR using degenerate oligonucleotide primers against conserved motifs in the VIb and VIII subdomains of the ACG family of serine/threonine protein kinases was utilised to amplify regions corresponding to the parasite homologue of the protein kinase A catalytic subunit. This putative protein kinase A fragment was used to isolate the entire gene from T. cruzi genomic libraries. The deduced 329 amino acid sequence of this gene contained all of the signature motifs of known protein kinase A catalytic subunit proteins. The recombinant protein expressed in Escherichia coli was shown to phosphorylate Kemptide, a synthetic peptide substrate of protein kinase A, in a protein kinase inhibitor (PKI)-inhibitory manner. Immunoprecipitation with polyclonal antisera raised against recombinant protein of this gene was able to pull-down PKI-inhibitory phosphotransferase activity from epimastigote lysates. Immunoblot and Northern blot analyses, in combination with enzyme activity assays, revealed that this gene was a stage-regulated enzyme in T. cruzi with higher levels and activity being present in epimastigotes compared with amastigotes or trypomastigotes. Overall these studies indicate that the cloned gene encodes an authentic protein kinase A catalytic subunit from T. cruzi and are the first demonstration of PKI-inhibitory phosphotransferase activity in an expressed protozoan protein kinase A catalytic subunit.

Novel molecules that interact with microtubules and have functional activity similar to Taxol.

Taxol is an antitumor drug approved by the FDA for the treatment of ovarian, breast and non-small-cell lung carcinomas. Originally isolated from the bark of the Pacific yew, Taxus brevifolia, it was the first natural product described that stabilized microtubules. In the past five years, a group of novel natural products, including the epothilones, discodermolide, eleutherobin, sarcodictyins and the laulimalides, all of which have biological activities similar to those of Taxol, has been discovered. In this review, we discuss each of these novel microtubule-stabilizing agents and the search for a common pharmacophore among them, taking into consideration recent advances in our understanding of the taxanes and tubulin.