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O John Semmes

Publications and source records attributed to O John Semmes.

At least 19 recordsLinked to original sources
PubMed2006

Site-specific phosphorylation differentiates active from inactive forms of the human T-cell leukemia virus type 1 Tax oncoprotein.

The human T-cell leukemia virus type 1 oncoprotein Tax is a phosphoprotein with a predominately nuclear subcellular localization that accomplishes multiple functions via protein-protein interactions. It has been proposed that regulation of this protein's pleiotropic functions may be accomplished through phosphorylation of specific amino acid residues. We have conducted a phosphoryl mapping of mammalian-expressed Tax protein using a combination of affinity purification, liquid chromatography tandem mass spectrometry, and site-directed substitution mutational analysis. We achieved physical coverage of 77% of the Tax sequence and identified four novel sites of phosphorylation at Thr-48, Thr-184, Thr-215, and Ser-336. Previously identified potential serine phosphorylation sites at Ser-10, Ser-77, and Ser-274 could not be confirmed by mass spectrometry. The functional significance of these novel phosphorylation events was evaluated by mutational analysis and subsequent evaluation for activity via both CREB and NF-kappaB-responsive promoters. Our results demonstrate that phosphorylation at Thr-215 is associated with loss of both Tax functions, phosphorylation at Thr-48 was specifically deficient for activation via NF-kappaB, and phosphorylation at Thr-184 and Ser-336 had no effect on these Tax functions. Semiquantitation of phosphopeptides revealed that the majority of Tax was phosphorylated at Thr-48, Thr-184, Thr-215, and Ser-336, whereas only a minor population of Tax was phosphorylated at either Ser-300 or Ser-301. These results suggest that both positive and negative phosphorylation signals result in the maintenance of a subfraction of Tax as "active" protein.

Amino Acid Sequence↗
PubMed2006

Lectin capture strategies combined with mass spectrometry for the discovery of serum glycoprotein biomarkers.

The application of mass spectrometry to identify disease biomarkers in clinical fluids like serum using high throughput protein expression profiling continues to evolve as technology development, clinical study design, and bioinformatics improve. Previous protein expression profiling studies have offered needed insight into issues of technical reproducibility, instrument calibration, sample preparation, study design, and supervised bioinformatic data analysis. In this overview, new strategies to increase the utility of protein expression profiling for clinical biomarker assay development are discussed with an emphasis on utilizing differential lectin-based glycoprotein capture and targeted immunoassays. The carbohydrate binding specificities of different lectins offer a biological affinity approach that complements existing mass spectrometer capabilities and retains automated throughput options. Specific examples using serum samples from prostate cancer and hepatocellular carcinoma subjects are provided along with suggested experimental strategies for integration of lectin-based methods into clinical fluid expression profiling strategies. Our example workflow incorporates the necessity of early validation in biomarker discovery using an immunoaffinity-based targeted analytical approach that integrates well with upstream discovery technologies.

Amino Acid Sequence↗
PubMed2006

Application of mass spectrometry to the discovery of biomarkers for detection of prostate cancer.

There has been an impressive emergence of mass spectrometry based technologies applied toward the study of proteins. Equally notable is the rapid adaptation of these technologies to biomedical approaches in the realm of clinical proteomics. Concerted efforts toward the elucidation of the proteomes of organ sites or specific disease state are proliferating and from these efforts come the promise of better diagnostics/prognostics and therapeutic intervention. Prostate cancer has been a focus of many such studies with the promise of improved care to patients via biomarkers derived from these proteomic approaches. The newer technologies provide higher analytical capabilities, employ automated liquid handling systems, fractionation techniques and bioinformatics tools for greater sensitivity and resolving power, more robust and higher throughput sample processing, and greater confidence in analytical results. In this prospects, we summarize the proteomic technologies applied to date in prostate cancer, along with their respective advantages and disadvantages. The development of newer proteomic strategies for use in future applications is also discussed.

Biomarkers, Tumor↗
PubMed2006

Human LINE-1 retrotransposon induces DNA damage and apoptosis in cancer cells.

BACKGROUND: Long interspersed nuclear elements (LINEs), Alu and endogenous retroviruses (ERVs) make up some 45% of human DNA. LINE-1 also called L1, is the most common family of non-LTR retrotransposons in the human genome and comprises about 17% of the genome. L1 elements require the integration into chromosomal target sites using L1-encoded endonuclease which creates staggering DNA breaks allowing the newly transposed L1 copies to integrate into the genome. L1 expression and retrotransposition in cancer cells might cause transcriptional deregulation, insertional mutations, DNA breaks, and an increased frequency of recombinations, contributing to genome instability. There is however little evidence on the mechanism of L1-induced genetic instability and its impact on cancer cell growth and proliferation. RESULTS: We report that L1 has genome-destabilizing effects indicated by an accumulation of gamma-H2AX foci, an early response to DNA strand breaks, in association with an abnormal cell cycle progression through a G2/M accumulation and an induction of apoptosis in breast cancer cells. In addition, we found that adjuvant L1 activation may lead to supra-additive killing when combined with radiation by enhancing the radiation lethality through induction of apoptosis that we have detected through Bax activation. CONCLUSION: L1 retrotransposition is sensed as a DNA damaging event through the creation DNA breaks involving L1-encoded endonuclease. The apparent synergistic interaction between L1 activation and radiation can further be utilized for targeted induction of cancer cell death. Thus, the role of retrotransoposons in general, and of L1 in particular, in DNA damage and repair assumes larger significance both for the understanding of mutagenicity and, potentially, for the control of cell proliferation and apoptosis.

Journal Article↗
PubMed2006

Deconvolution filters to enhance resolution of dense time-of-flight survey spectra in the time-lag optimization range.

By applying time-domain filters to time-of-flight (TOF) mass spectrometry signals, we have simultaneously smoothed and narrowed spectra resulting in improved resolution and increased signal-to-noise ratios. This filtering procedure has an advantage over detailed curve fitting of spectra in the case of large dense spectra, when neither the location nor the number of mass peaks is known a priori. This time series method is directly applicable in the time lag optimization range, where point density per peak is constant. We present a systematic methodology to optimize the filters according to any desired figure of merit, illustrating the procedure by optimizing the signal-to-noise per unit bandwidth of matrix-assisted laser desorption/ionization (MALDI) data. We also introduce a nonlinear filter that reduces the spurious structure that often accompanies deconvolution filters. The net result of the application of these filters is that we can identify new structures in dense MALDI-TOF data, clearly showing small adducts to heavy biomolecules.

Algorithms↗
PubMed2006

Resampling and deconvolution of linear time-of-flight records for enhanced protein profiling.

We have developed a peak deconvolution strategy that is applicable to the full mass range of a time-of-flight (TOF) spectrum. This strategy involves resampling a spectrum to create a time series that has equal peak widths (in time) across the entire spectrum, and then using the deconvolution filters we have previously described. We use this technique to deconvolve the protein mass spectra for blood serum and cell lysates acquired on three separate TOF instruments. Following deconvolution, we resolve spectral structures consistent with expected events such as multiply charged ions, matrix adducts and post-translational protein modifications. The deconvolution procedure produces a 40% improvement in the resolution and enhanced experimental sensitivity over the full length of the linear TOF record, up to m/z 150 000. This approach is particularly appropriate for automated data analysis and peak detection in dense TOF spectra.

Algorithms↗
PubMed2006

Adult T cell leukemia: a tale of two T cells.

Human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent for the development of an aggressive hematologic neoplasia termed adult T cell leukemia/lymphoma (ATLL). Although the virus infects T cell subsets that display either CD4 or CD8 cell surface markers, the leukemic cell is exclusively of the CD4+ subtype. In the article by Sibon et al. in this issue of the JCI, the authors demonstrate that the molecular basis for clonal expansion differs between these 2 infected T cell populations (see the related article beginning on page 974). The molecular events associated with a preleukemic state, such as genomic instability, polynucleation, and cell cycle redistribution, were only observed in CD4+ T cells. This finding provides a molecular-based mechanism for the restriction of the leukemic phenotype to the CD4+ T cell subtype.

Adult↗
PubMed2005

PDZ binding motif of HTLV-1 Tax promotes virus-mediated T-cell proliferation in vitro and persistence in vivo.

HTLV-1 cellular transformation and disease induction is dependent on expression of the viral Tax oncoprotein. PDZ is a modular protein interaction domain used in organizing signaling complexes in eukaryotic cells through recognition of a specific binding motif in partner proteins. Tax-1, but not Tax-2, contains a PDZ-binding domain motif (PBM) that promotes the interaction with several cellular PDZ proteins. Herein, we investigate the contribution of the Tax-1 PBM in HTLV-induced proliferation and immortalization of primary T cells in vitro and viral survival in an infectious rabbit animal model. We generated several HTLV-1 and HTLV-2 Tax viral mutants, including HTLV-1deltaPBM, HTLV-2+C22(+PBM), and HTLV-2+ C18(deltaPBM). All Tax mutants maintained the ability to significantly activate the CREB/ATF or NFkappaB signaling pathways. Microtiter proliferation assays revealed that the Tax-1 PBM significantly increases both HTLV-1- and HTLV-2-induced primary T-cell proliferation. In addition, Tax-1 PBM was responsible for the micronuclei induction activity of Tax-1 relative to that of Tax-2. Viral infection and persistence were severely attenuated in rabbits inoculated with HTLV-1deltaPBM. Our results provide the first direct evidence suggesting that PBM-mediated associations between Tax-1 and cellular proteins play a key role in HTLV-induced cell proliferation and genetic instability in vitro and facilitate viral persistence in vivo.

Amino Acid Motifs↗
PubMed2005

Functional clustering algorithm for high-dimensional proteomics data.

Clustering proteomics data is a challenging problem for any traditional clustering algorithm. Usually, the number of samples is largely smaller than the number of protein peaks. The use of a clustering algorithm which does not take into consideration the number of features of variables (here the number of peaks) is needed. An innovative hierarchical clustering algorithm may be a good approach. We propose here a new dissimilarity measure for the hierarchical clustering combined with a functional data analysis. We present a specific application of functional data analysis (FDA) to a high-throughput proteomics study. The high performance of the proposed algorithm is compared to two popular dissimilarity measures in the clustering of normal and human T-cell leukemia virus type 1 (HTLV-1)-infected patients samples.

Journal Article↗
PubMed2005

Serum levels of an isoform of apolipoprotein A-II as a potential marker for prostate cancer.

PURPOSE: We recently showed that protein expression profiling of serum using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) has potential as a diagnostic approach for detection of prostate cancer. As a parallel effort, we have been pursuing the identification of the protein(s) comprising the individual discriminatory "peaks" and evaluating their utility as potential biomarkers for prostate disease. EXPERIMENTAL DESIGN: We employed liquid chromatography, gel electrophoresis and tandem mass spectroscopy to isolate and identify a protein that correlates with observed SELDI-TOF MS mass/charge (m/z) values. Immunodepletion, immunoassay, and Western analysis were used to verify that the identified protein generated the observed SELDI peak. Subsequent immunohistochemistry was used to examine the expression of the proteins in prostate tumors. RESULTS: An 8,946 m/z SELDI-TOF MS peak was found to retain discriminatory value in each of two separate data sets with an increased expression in the diseased state. Sequence identification by liquid chromatography-MS/MS and subsequent immunoassays verified that an isoform of apolipoprotein A-II (ApoA-II) is the observed 8,946 m/z SELDI peak. Immunohistochemistry revealed that ApoA-II is overexpressed in prostate tumors. SELDI-based immunoassay revealed that an 8.9-kDa isoform of ApoA-II is specifically overexpressed in serum from individuals with prostate cancer. ApoA-II was also overexpressed in the serum of individuals with prostate cancer who have normal prostate-specific antigen (0-4.0 ng/mL). CONCLUSIONS: We have identified an isoform of ApoA-II giving rise to an 8.9K m/z SELDI "peak" that is specifically overexpressed in prostate disease. The ability of ApoA-II to detect disease in patients with normal prostate-specific antigen suggests potential utility of the marker in identifying indolent disease.

Apolipoprotein A-II↗
PubMed2005

SELDI-TOF MS profiling of serum for detection of the progression of chronic hepatitis C to hepatocellular carcinoma.

Proteomic profiling of serum is an emerging technique to identify new biomarkers indicative of disease severity and progression. The objective of our study was to assess the use of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to identify multiple serum protein biomarkers for detection of liver disease progression to hepatocellular carcinoma (HCC). A cohort of 170 serum samples obtained from subjects in the United States with no liver disease (n = 39), liver diseases not associated with cirrhosis (n = 36), cirrhosis (n = 38), or HCC (n = 57) were applied to metal affinity protein chips for protein profiling by SELDI-TOF MS. Across the four test groups, 38 differentially expressed proteins were used to generate multiple decision classification trees to distinguish the known disease states. Analysis of a subset of samples with only hepatitis C virus (HCV)-related disease was emphasized. The serum protein profiles of control patients were readily distinguished from each HCV-associated disease state. Two-way comparisons of chronic hepatitis C, HCV cirrhosis, or HCV-HCC versus healthy had a sensitivity/specificity range of 74% to 95%. For distinguishing chronic HCV from HCV-HCC, a sensitivity of 61% and a specificity of 76% were obtained. However, when the values of known serum markers alpha fetoprotein, des-gamma carboxyprothrombin, and GP73 were combined with the SELDI peak values, the sensitivity and specifity improved to 75% and 92%, respectively. In conclusion, SELDI-TOF MS serum profiling is able to distinguish HCC from liver disease before cirrhosis as well as cirrhosis, especially in patients with HCV infection compared with other etiologies.

Adult↗
PubMed2005

Analysis of Human Proteome Organization Plasma Proteome Project (HUPO PPP) reference specimens using surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry: multi-institution correlation of spectra and identification of biomarkers.

We report on a multicenter analysis of HUPO reference specimens using SELDI-TOF MS. Eight sites submitted data obtained from serum and plasma reference specimen analysis. Spectra from five sites passed preliminary quality assurance tests and were subjected to further analysis. Intralaboratory CVs varied from 15 to 43%. A correlation coefficient matrix generated using data from these five sites demonstrated high level of correlation, with values >0.7 on 37 of 42 spectra. More than 50 peaks were differentially present among the various sample types, as observed on three chip surfaces. Additionally, peaks at approximately 9200 and approximately 15,950 m/z were present only in select reference specimens. Chromatographic fractionation using anion-exchange, membrane cutoff, and reverse phase chromatography, was employed for protein purification of the approximately 9200 m/z peak. It was identified as the haptoglobin alpha subunit after peptide mass fingerprinting and high-resolution MS/MS analysis. The differential expression of this protein was confirmed by Western blot analysis. These pilot studies demonstrate the potential of the SELDI platform for reproducible and consistent analysis of serum/plasma across multiple sites and also for targeted biomarker discovery and protein identification. This approach could be exploited for population-based studies in all phases of the HUPO PPP.

Biomarkers↗
PubMed2005

The bZIP transcription factor ATFx binds human T-cell leukemia virus type 1 (HTLV-1) Tax and represses HTLV-1 long terminal repeat-mediated transcription.

The human T-cell leukemia virus type 1 (HTLV-1) viral protein Tax is a transactivator of transcription driven by the cognate viral long terminal repeat (LTR). Tax exerts its effect through three nonidentical copies of the Tax-responsive element (TxRE), a member of the asymmetric cyclic AMP response element (CRE) family of enhancer sequences. Transactivation is mediated via interaction of Tax with members of the CREB/ATF family bound to TxRE. We have identified a cellular repressor of transcription, activating transcription factor x (ATFx), as a novel Tax-binding protein. In addition to binding directly to Tax we show by electrophoretic mobility shift assay that ATFx binds to the TxRE enhancer element via the bZIP domain. The functional impact of this bridging interaction results in repression of both basal and Tax-induced transcription from the HTLV-1 LTR. ATFx is unique among ATF family of proteins in that it is cell cycle regulated and exerts a tight repressive control over apoptotic signaling. We propose that recruitment of ATFx to the HTLV-1 LTR serves to link viral transcription with critical events in cellular homeostasis.

Activating Transcription Factors↗
PubMed2005

Evaluation of serum protein profiling by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry for the detection of prostate cancer: I. Assessment of platform reproducibility.

BACKGROUND: Protein expression profiling for differences indicative of early cancer has promise for improving diagnostics. This report describes the first stage of a National Cancer Institute/Early Detection Research Network-sponsored multiinstitutional evaluation and validation of this approach for detection of prostate cancer. METHODS: Two sequential experimental phases were conducted to establish interlaboratory calibration and standardization of the surface-enhanced laser desorption (SELDI) instrumental and assay platform output. We first established whether the output from multiple calibrated Protein Biosystem II SELDI-ionization time-of-flight mass spectrometry (TOF-MS) instruments demonstrated acceptable interlaboratory reproducibility. This was determined by measuring mass accuracy, resolution, signal-to-noise ratio, and normalized intensity of three m/z "peaks" present in a standard pooled serum sample. We next evaluated the ability of the calibrated and standardized instrumentation to accurately differentiate between selected cases of prostate cancer and control by use of an algorithm developed from data derived from a single site 2 years earlier. RESULTS: When the described standard operating procedures were established at all laboratory sites, the across-laboratory measurements revealed a CV for mass accuracy of 0.1%, signal-to-noise ratio of approximately 40%, and normalized intensity of 15-36% for the three pooled serum peaks. This was comparable to the intralaboratory measurements of the same peaks. The instrument systems were then challenged with sera from a selected group of 14 cases and 14 controls. The classification agreement between each site and the established decision algorithm were examined by use of both raw peak intensity boosting and ranked peak intensity boosting. All six sites achieved perfect blinded classification for all samples when boosted alignment of raw intensities was used. Four of six sites achieved perfect blinded classification with ranked intensities, with one site passing the criteria of 26 of 28 correct and one site failing with 19 of 28 correct. CONCLUSIONS: These results demonstrate that "between-laboratory" reproducibility of SELDI-TOF-MS serum profiling approaches that of "within-laboratory" reproducibility as determined by measuring discrete m/z peaks over time and across laboratories.

Calibration↗
PubMed2005

Serum, salivary and tissue proteomics for discovery of biomarkers for head and neck cancers.

Initial clinically oriented applications of emerging proteomic technologies that aim to identify biomarkers for head and neck squamous cell carcinoma diagnostics have yielded promising results. The development of new proteomic diagnostics remains critical for the early detection of head and neck squamous cell carcinoma at more treatable stages. Prognostic markers for disease recurrence and treatment sensitivities are also required. In this overview of current biomarker identification strategies for head and neck squamous cell carcinoma, different combinations of mass spectrometry platforms, laser capture microscopy and 2D gel electrophoresis procedures are summarized as applied to readily available clinical specimens (tissue, blood and saliva). Issues related to assay reproducibility, management of large data sets and future improvements in clinical proteomics are also addressed.

Algorithms↗
PubMed2004

Enhancement of sensitivity and resolution of surface-enhanced laser desorption/ionization time-of-flight mass spectrometric records for serum peptides using time-series analysis techniques.

BACKGROUND: Measurement of peptide/protein concentrations in biological samples for biomarker discovery commonly uses high-sensitivity mass spectrometers with a surface-processing procedure to concentrate the important peptides. These time-of-flight (TOF) instruments typically have low mass resolution and considerable electronic noise associated with their detectors. The net result is unnecessary overlapping of peaks, apparent mass jitter, and difficulty in distinguishing mass peaks from background noise. Many of these effects can be reduced by processing the signal using standard time-series background subtraction, calibration, and filtering techniques. METHODS: Surface-enhanced laser desorption/ionization (SELDI) spectra were acquired on a PBS II instrument from blank, hydrophobic, and IMAC-Cu ProteinChip arrays (Ciphergen Biosystems, Inc.) incubated with calibration peptide mixtures or pooled serum. TOF data were recorded after single and multiple laser shots at different positions. Correlative analysis was used for time-series calibration. Target filters were used to suppress noise and enhance resolution after baseline removal and noise rescaling. RESULTS: The developed algorithms compensated for the electronic noise attributable to detector overload, removed the baseline caused by charge accumulation, detected and corrected mass peak jitter, enhanced signal amplitude at higher masses, and improved the resolution by using a deconvolution filter. CONCLUSIONS: These time-series techniques, when applied to SELDI-TOF data before any peak identification procedure, can improve the data to make the peak identification process simpler and more robust. These improvements may be applicable to most TOF instrumentation that uses analog (rather than counting) detectors.

Artifacts↗
PubMed2004

Surfaced-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) differentiation of serum protein profiles of BRCA-1 and sporadic breast cancer.

BACKGROUND: BRCA-1 mutations predispose women to early onset breast cancer, but approximately 20% never develop cancer. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) profiling can differentiate protein signatures of cancer and normal subjects. Our objective was to distinguish women with BRCA-1 mutations who developed breast cancer (BRCA-1 Ca) from those who did not (Carrier), normal volunteers (NL), and women with sporadic breast cancer (SBC), using SELDI-TOF. METHODS: Baseline serum specimens were obtained from women with BRCA-1 mutations without cancer, SBC, and NL. BRCA-1 women were later divided into two cohorts, pending cancer development. The sera were spotted onto protein chips for SELDI-TOF analysis and analyzed with classification algorithm software. RESULTS: BRCA-1 Ca patients (n = 15) developed cancer within 3 years of baseline, while BRCA-1 carriers (n = 15) were cancer-free in 7 years of follow-up. SELDI-TOF analysis revealed differentially expressed proteins (P < .05) between BRCA-1 Ca, Carrier, and SBC patients (n = 16), such that 13/15 BRCA-1 Ca vs. Carrier women were correctly identified (sensitivity/specificity of 87%/87%) and 14/15 BRCA-1 Ca vs. SBC patients were correctly identified (sensitivity/specificity 94%/100%). Profiles of Carriers resembled NL profiles (n = 16). CONCLUSIONS: SELDI-TOF protein profiles from this small pilot study distinguished between women with BRCA-1 Ca, Carriers, and women with SBC. Whether BRCA-1 Ca represents earlier detection of occult cancer or other risk factors is unknown. Follow-up studies with larger numbers and longer follow-up are required to validate these findings but may allow more timely prophylactic or therapeutic strategies.

Adult↗