A new biomarker panel in bronchoalveolar lavage for an improved lung cancer diagnosis.

INTRODUCTION: The enormous biological complexity and high mortality rate of lung cancer highlights the need for new global approaches for the discovery of reliable early diagnostic biomarkers. The study of bronchoalveolar lavage samples by proteomic techniques could identify new lung cancer biomarkers and may provide promising noninvasive diagnostic tools able to enhance the sensitivity of current methods. METHODS: First, an observational prospective study was designed to assess protein expression differences in bronchoalveolar lavages from patients with (n = 139) and without (n = 49) lung cancer, using two-dimensional gel electrophoresis and subsequent protein identification by mass spectrometry. Second, validation of candidate biomarkers was performed by bead-based immunoassays with a different patient cohort (204 patients, 48 controls). RESULTS: Thirty-two differentially expressed proteins were identified in bronchoalveolar lavages, 10 of which were confirmed by immunoassays. The expression levels of APOA1, CO4A, CRP, GSTP1, and SAMP led to a lung cancer diagnostic panel that reached 95% sensitivity and 81% specificity, and the quantification of STMN1 and GSTP1 proteins allowed the two main lung cancer subtypes to be discriminated with 90% sensitivity and 57% specificity. CONCLUSIONS: Bronchoalveolar lavage represents a promising noninvasive source of lung cancer specific protein biomarkers with high diagnostic accuracy. Measurement of APOA1, CO4A, CRP, GSTP1, SAMP, and STMN1 in this fluid may be a useful tool for lung cancer diagnosis, although a further validation in a larger clinical set is required for early stages.

Reconstitution in liposome bilayers enhances nucleotide binding affinity and ATP-specificity of TrwB conjugative coupling protein.

Bacterial conjugative systems code for an essential membrane protein that couples the relaxosome to the DNA transport apparatus, called type IV coupling protein (T4CP). TrwB is the T4CP of the conjugative plasmid R388. In earlier work we found that this protein, purified in the presence of detergents, binds preferentially purine nucleotides trisphosphate. In contrast a soluble truncated mutant TrwBΔN70 binds uniformly all nucleotides tested. In this work, TrwB has been successfully reconstituted into liposomes. The non-membranous portion of the protein is almost exclusively oriented towards the outside of the vesicles. Functional analysis of TrwB proteoliposomes demonstrates that when the protein is inserted into the lipid bilayer the affinity for adenine and guanine nucleotides is enhanced as compared to that of the protein purified in detergent or to the soluble deletion mutant, TrwBΔN70. The protein specificity for adenine nucleotides is also increased. No ATPase activity has been found in TrwB reconstituted in proteoliposomes. This result suggests that the N-terminal transmembrane segment of this T4CP interferes with its ATPase activity and can be taken to imply that the TrwB transmembrane domain plays a regulatory role in its biological activity.

Comprehensive genomic screens identify a role for PLZF-RARalpha as a positive regulator of cell proliferation via direct regulation of c-MYC.

The t(11;17)(q23;q21) translocation is associated with a retinoic acid (RA)-insensitive form of acute promyelocytic leukemia (APL), involving the production of reciprocal fusion proteins, promyelocytic leukemia zinc finger-retinoic acid receptor alpha (PLZF-RARalpha) and RARalpha-PLZF. Using a combination of chromatin immunoprecipitation promotor arrays (ChIP-chip) and gene expression profiling, we identify novel, direct target genes of PLZF-RARalpha that tend to be repressed in APL compared with other myeloid leukemias, supporting the role of PLZF-RARalpha as an aberrant repressor in APL. In primary murine hematopoietic progenitors, PLZF-RARalpha promotes cell growth, and represses Dusp6 and Cdkn2d, while inducing c-Myc expression, consistent with its role in leukemogenesis. PLZF-RARalpha binds to a region of the c-MYC promoter overlapping a functional PLZF site and antagonizes PLZF-mediated repression, suggesting that PLZF-RARalpha may act as a dominant-negative version of PLZF by affecting the regulation of shared targets. RA induced the differentiation of PLZF-RARalpha-transformed murine hematopoietic cells and reduced the frequency of clonogenic progenitors, concomitant with c-Myc down-regulation. Surviving RA-treated cells retained the ability to be replated and this was associated with sustained c-Myc expression and repression of Dusp6, suggesting a role for these genes in maintaining a self-renewal pathway triggered by PLZF-RARalpha.

Chromatin modulation by oncogenic transcription factors: new complexity, new therapeutic targets.

Oncogenic transcription factors such as PML-RARalpha, RUNX1-MTG8, and others work in large part by the recruitment of inhibitors of gene transcription to target promoters leading to aberrant repression of gene expression. PML-RARalpha, an archetypal chimeric oncoprotein, was previously shown to bring complexes of histone deacetylases (HDACs), histone methyltransferases (HMTases), and DNA methyl transferases (DNMTs) to target genes. In this issue of Cancer Cell, Villa et al. show that the full complement of chromatin machinery can be commandeered by these transcription factors with the polycomb group of proteins representing the newest identified recruit.

The transmembrane domain provides nucleotide binding specificity to the bacterial conjugation protein TrwB.

In order to understand the functional significance of the transmembrane domain of TrwB, an integral membrane protein involved in bacterial conjugation, the protein was purified in the native, and also as a truncated soluble form (TrwBDeltaN70). The intact protein (TrwB) binds preferentially purine over pyrimidine nucleotides, NTPs over NDPs, and ribo- over deoxyribonucleotides. In contrast, TrwBDeltaN70 binds uniformly all tested nucleotides. The transmembrane domain has the general effect of making the nucleotide binding site(s) less accessible, but more selective. This is in contrast to other membrane proteins in which most of the protein mass, including the catalytic domain, is outside the membrane, but whose activity is not modified by the presence or absence of the transmembrane segment.

Histone acetyltransferase activity of p300 is required for transcriptional repression by the promyelocytic leukemia zinc finger protein.

Histone acetyltransferase (HAT) activities of proteins such as p300, CBP, and P/CAF play important roles in activation of gene expression. We now show that the HAT activity of p300 can also be required for down-regulation of transcription by a DNA binding repressor protein. Promyelocytic leukemia zinc finger (PLZF), originally identified as a fusion with retinoic acid receptor alpha in rare cases of all-trans-retinoic acid-resistant acute promyelocytic leukemia, is a transcriptional repressor that recruits histone deacetylase-containing corepressor complexes to specific DNA binding sites. PLZF associates with p300 in vivo, and its ability to repress transcription is specifically dependent on HAT activity of p300 and acetylation of lysines in its C-terminal C2-H2 zinc finger motif. An acetylation site mutant of PLZF does not repress transcription and is functionally deficient in a colony suppression assay despite retaining its abilities to interact with corepressor/histone deacetylase complexes. This is due to the fact that acetylation of PLZF activates its ability to bind specific DNA sequences both in vitro and in vivo. Taken together, our results indicate that a histone deacetylase-dependent transcriptional repressor can be positively regulated through acetylation and point to an unexpected role of a coactivator protein in transcriptional repression.

Role of the transmembrane domain in the stability of TrwB, an integral protein involved in bacterial conjugation.

TrwB is an integral membrane protein encoded by the conjugative plasmid R388. TrwB binds ATP and is essential for R388-directed bacterial conjugation. The protein consists of a cytosolic domain, which contains an ATP-binding site, and a transmembrane domain. The complete protein has been purified in the presence of detergents, and in addition, the cytosolic domain has also been isolated in the form of a soluble truncated protein, TrwBDeltaN70. The availability of intact and truncated forms of the protein provides a convenient system to study the role of the transmembrane domain in the stability of TrwB. Protein denaturation was achieved by heat, in the presence of guanidinium HCl, or under low salt conditions. In all three cases TrwB was significantly more stable than TrwBDeltaN70 with other conditions being the same. IR spectroscopy of the native and truncated forms revealed significant differences between them. In addition, it was found that TrwBDeltaN70 was stabilized in dispersions of non-ionic detergent, suggesting the presence of hydrophobic patches on the surface of the truncated protein. IR spectroscopy also confirmed the conformational stability provided by the detergent. These results suggest that in integral membrane proteins consisting of a transmembrane and a cytosolic domain, the transmembrane portion may have a role beyond the mere anchoring of the protein to the cell membrane. In addition, this study indicates that the truncated soluble parts of two-domain membrane proteins may not reflect the physiological conformation of their native counterparts.

Purification and properties of TrwB, a hexameric, ATP-binding integral membrane protein essential for R388 plasmid conjugation.

TrwB is an integral membrane protein linking the relaxosome to the DNA transport apparatus in plasmid R388 conjugation. Native TrwB has been purified in monomeric and hexameric forms, in the presence of dodecylmaltoside from overexpressing bacterial cells. A truncated protein (TrwBDeltaN70) that lacked the transmembrane domain could be purified only in the monomeric form. Electron microscopy images revealed the hexameric structure and were in fact superimposable to the previously published atomic structure for TrwBDeltaN70. In addition, the electron micrographs showed an appendix, approximately 25 A wide, corresponding to the transmembrane region of TrwB. TrwB was located in the bacterial inner membrane in agreement with its proposed coupling role. Purified TrwB hexamers and monomers bound tightly the fluorescent ATP analogue TNP-ATP. A mutant in the Walker A motif, TrwB-K136T, was equally purified and found to bind TNP-ATP with a similar affinity to that of the wild type. However, the TNP-ATP affinity of TrwBDeltaN70 was significantly reduced in comparison with the TrwB hexamers. Competition experiments in which ATP was used to displace TNP-ATP gave an estimate of ATP binding by TrwB (K(d)((ATP)) = 0.48 mm for hexamers). The transmembrane domain appears to be involved in TrwB protein hexamerization and also influences its nucleotide binding properties.