Hundreds of candidate 14-3-3-binding (phospho)proteins have been reported in publications that describe one interaction at a time, as well as high-throughput 14-3-3-affinity and mass spectrometry-based studies. Here, we transcribed these data into a common format, deposited the collated data from low-throughput studies in MINT (http://mint.bio.uniroma2.it/mint), and compared the low- and high-throughput data in VisANT graphs that are easy to analyze and extend. Exploring the graphs prompted questions about technical and biological specificity, which were addressed experimentally, resulting in identification of phosphorylated 14-3-3-binding sites in the mitochondrial import sequence of the iron-sulfur cluster assembly enzyme (ISCU), cytoplasmic domains of the mitochondrial fission factor (MFF), and endoplasmic reticulum-tethered receptor expression-enhancing protein 4 (REEP4), RNA regulator SMAUG2, and cytoskeletal regulatory proteins, namely debrin-like protein (DBNL) and kinesin light chain (KLC) isoforms. Therefore, 14-3-3s undergo physiological interactions with proteins that are destined for diverse subcellular locations. Graphing and validating interactions underpins efforts to use 14-3-3-phosphoproteomics to identify mechanisms and biomarkers for signaling pathways in health and disease.
We sought to analyze the indications and outcome of extracorporeal membrane oxygenation (ECMO) for early primary graft failure and determine its impact on long-term graft function and rejection risk.
Early post-operative graft failure requiring ECMO can complicate heart transplantation.
A retrospective review of all children requiring ECMO in the early period after transplantation from 1990 to 2007 was undertaken.
Twenty-eight (9%) of 310 children who underwent transplantation for cardiomyopathy (n = 5) or congenital heart disease (n = 23) required ECMO support. The total ischemic time was significantly longer for ECMO-rescued recipients compared with our overall transplantation population (276 ± 86 min vs. 242 ± 70 min, p < 0.01). The indication for transplantation, for ECMO support, and the timing of cannulation had no impact on survival. Hyperacute rejection was uncommon. Fifteen children were successfully weaned off ECMO and discharged alive (54%). Mean duration of ECMO was 2.8 days for survivors (median 3 days) compared with 4.8 days for nonsurvivors (median 5 days). There was 100% 3-year survival in the ECMO survivor group, with 13 patients (46%) currently alive at a mean follow-up of 8.1 ± 3.8 years. The graft function was preserved (shortening fraction 36 ± 7%), despite an increased number of early rejection episodes (1.7 ± 1.6 vs. 0.7 ± 1.3, overall transplant population, p < 0.05) and hemodynamically comprising rejection episodes (1.3 ± 1.9 vs. 0.7 ± 1.3, overall transplant population, p < 0.05).
Overall survival was 54%, with all patients surviving to at least 3 years after undergoing transplantation. None of the children requiring >4 days of ECMO support survived. Despite an increased number of early and hemodynamically compromising rejections, the long-term graft function is similar to our overall transplantation population.
extracorporeal membrane oxygenation; child; heart transplantation; right-sided heart failure; left-sided heart failure; hypertension pulmonary
Incision into the ventricular septum in complex biventricular repair is controversial, and has been blamed for impairing left ventricular function. This retrospective study evaluates the risk of a ventricular septal incision in patients undergoing double outlet right ventricle (DORV) repair and Ross–Konno procedure.
From January 2003 to September 2007, 11 patients with DORV had a ventricular septum (VS) incision and 12 DORV patients did not. Sixteen patients had a Ross–Konno, and 16 had an isolated Ross procedure. The ventricular septal incision was made to match at least the diameter of a normal aortic annulus. In DORV, the VSD was enlarged superiorly and to the left. In the Ross–Konno, the aortic annulus was enlarged towards the septum posteriorly and to the left.
The median follow-up for the study is 19 months (1 month–4 years). For DORV, there were no significant differences in discharge mortality ( p = 0.22), late mortality ( p = 0.48), or late mortality plus heart transplant ( p = 0.093). Although patients with DORV and VSD enlargement have a more complex postoperative course, there were no differences in ECMO use ( p = 0.093), occurrence of permanent AV block ( p = 0.55), left ventricular ejection fraction (LVEF) ( p = 0.40), or shortening fraction (LVSF) ( p = 0.50). Similarly, for the Ross–Konno there were no significant differences in discharge mortality ( p = 0.30), late mortality ( p = NS), LVEF (p = 0.90) and LVSF ( p = 0.52) compared to the Ross, even though the Ross–Konno patients were significantly younger ( p < 0.0001).
Making a ventricular septal incision in DORV repair and in the Ross–Konno operation does not increase mortality and does not impair the LV function. The restriction of the VSD remains an important issue in the management of complex DORV. These encouraging results need to be confirmed by larger series.
Double outlet right ventricle; Ventricular septal defect; Ross–Konno
The purpose of this study was to describe the long-term outcome of infants with hypoplastic left heart syndrome (HLHS) who underwent placement of internal pulmonary artery bands as part of a transcatheter palliation procedure followed by primary heart transplantation. Transcatheter palliation included stenting of the ductus arteriosus, decompression of the left atrium by atrial septostomy, and internal pulmonary artery band placement. Cardiac hemodynamics, pulmonary artery architecture, and pulmonary artery growth since transplantation are described. Nine infants with HLHS had internal pulmonary artery bands placed and underwent successful heart transplant. No infant required reconstruction of the pulmonary arteries at the time of transplant. At 1 year after transplant, all of the recipients had normal mean pulmonary artery pressure, pulmonary vascular resistance, and transpulmonary gradient. Pulmonary angiography performed at 1 year after transplant demonstrated no distortion of pulmonary artery anatomy with significant interval growth of the branch pulmonary arteries. There was 100% survival to hospital discharge after transplant in this cohort of infants. Transcatheter placement of internal pulmonary artery bands for HLHS offers protection of the pulmonary vascular bed while preserving pulmonary artery architecture and growth with good long-term outcome.
Congenital heart disease; Heart catheterization; Heart transplant; Infant
The genus Perkinsus occupies a precarious phylogenetic position. To gain a better understanding of the relationship between perkinsids, dinoflagellates and other alveolates, we analyzed the nuclear-encoded spliced-leader (SL) RNA and mitochondrial genes, intron prevalence, and multi-protein phylogenies. In contrast to the canonical 22-nt SL found in dinoflagellates (DinoSL), P. marinus has a shorter (21-nt) and a longer (22-nt) SL with slightly different sequences than DinoSL. The major SL RNA transcripts range in size between 80–83 nt in P. marinus, and ∼83 nt in P. chesapeaki, significantly larger than the typical ≤56-nt dinoflagellate SL RNA. In most of the phylogenetic trees based on 41 predicted protein sequences, P. marinus branched at the base of the dinoflagellate clade that included the ancient taxa Oxyrrhis and Amoebophrya, sister to the clade of apicomplexans, and in some cases clustered with apicomplexans as a sister to the dinoflagellate clade. Of 104 Perkinsus spp. genes examined 69.2% had introns, a higher intron prevalence than in dinoflagellates. Examination of Perkinsus spp. mitochondrial cytochrome B and cytochrome C oxidase subunit I genes and their cDNAs revealed no mRNA editing, but these transcripts can only be translated when frameshifts are introduced at every AGG and CCC codon as if AGGY codes for glycine and CCCCU for proline. These results, along with the presence of the numerous uncharacterized ‘marine alveolate group I' and Perkinsus-like lineages separating perkinsids from core dinoflagellates, expand support for the affiliation of the genus Perkinsus with an independent lineage (Perkinsozoa) positioned between the phyla of Apicomplexa and Dinoflagellata.
The phosphorylation and dephosphorylation of proteins by kinases and phosphatases constitute an essential regulatory network in eukaryotic cells. This network supports the flow of information from sensors through signaling systems to effector molecules, and ultimately drives the phenotype and function of cells, tissues, and organisms. Dysregulation of this process has severe consequences and is one of the main factors in the emergence and progression of diseases, including cancer. Thus, major efforts have been invested in developing specific inhibitors that modulate the activity of individual kinases or phosphatases; however, it has been difficult to assess how such pharmacological interventions would affect the cellular signaling network as a whole. Here, we used label-free, quantitative phosphoproteomics in a systematically perturbed model organism (Saccharomyces cerevisiae) to determine the relationships between 97 kinases, 27 phosphatases, and more than 1000 phosphoproteins. We identified 8814 regulated phosphorylation events, describing the first system-wide protein phosphorylation network in vivo. Our results show that, at steady state, inactivation of most kinases and phosphatases affected large parts of the phosphorylation-modulated signal transduction machinery, and not only the immediate downstream targets. The observed cellular growth phenotype was often well maintained despite the perturbations, arguing for considerable robustness in the system. Our results serve to constrain future models of cellular signaling and reinforce the idea that simple linear representations of signaling pathways might be insufficient for drug development and for describing organismal homeostasis.
Since its inception, proteomics has essentially operated in a discovery mode with the goal of identifying and quantifying the maximal number of proteins in a sample. Increasingly, proteomic measurements are also supporting hypothesis-driven studies, in which a predetermined set of proteins is consistently detected and quantified in multiple samples. Selected reaction monitoring (SRM) is a targeted mass spectrometric technique that supports the detection and quantification of specific proteins in complex samples at high sensitivity and reproducibility. Here, we describe ATAQS, an integrated software platform that supports all stages of targeted, SRM-based proteomics experiments including target selection, transition optimization and post acquisition data analysis. This software will significantly facilitate the use of targeted proteomic techniques and contribute to the generation of highly sensitive, reproducible and complete datasets that are particularly critical for the discovery and validation of targets in hypothesis-driven studies in systems biology.
We introduce a new open source software pipeline, ATAQS (Automated and Targeted Analysis with Quantitative SRM), which consists of a number of modules that collectively support the SRM assay development workflow for targeted proteomic experiments (project management and generation of protein, peptide and transitions and the validation of peptide detection by SRM). ATAQS provides a flexible pipeline for end-users by allowing the workflow to start or end at any point of the pipeline, and for computational biologists, by enabling the easy extension of java algorithm classes for their own algorithm plug-in or connection via an external web site.
This integrated system supports all steps in a SRM-based experiment and provides a user-friendly GUI that can be run by any operating system that allows the installation of the Mozilla Firefox web browser.
Targeted proteomics via SRM is a powerful new technique that enables the reproducible and accurate identification and quantification of sets of proteins of interest. ATAQS is the first open-source software that supports all steps of the targeted proteomics workflow. ATAQS also provides software API (Application Program Interface) documentation that enables the addition of new algorithms to each of the workflow steps. The software, installation guide and sample dataset can be found in http://tools.proteomecenter.org/ATAQS/ATAQS.html
We present the case of a patient with intraoperative femoral fracture during THA, which was repaired using cerclage fixation and insertion of an hydroxyapatite-coated cementless stem. The patient was evaluated postoperatively using radiostereometry during a 2-year course, and despite a large amount of subsidence and rotation, stabilization occurred and was maintained by 6 months. By evaluating the pattern of stem migration after intraoperative fracture, this case shows, even in the presence of instability, a successful clinical outcome can be achieved using an hydroxyapatite-coated cementless stem.
The 5′ cap of human messenger RNA consists of an inverted 7-methylguanosine linked to the first transcribed nucleotide by a unique 5′–5′ triphosphate bond followed by 2′-O-ribose methylation of the first and often the second transcribed nucleotides, likely serving to modify efficiency of transcript processing, translation and stability. We report the validation of a human enzyme that methylates the ribose of the second transcribed nucleotide encoded by FTSJD1, henceforth renamed HMTR2 to reflect function. Purified recombinant hMTr2 protein transfers a methyl group from S-adenosylmethionine to the 2′-O-ribose of the second nucleotide of messenger RNA and small nuclear RNA. Neither N7 methylation of the guanosine cap nor 2′-O-ribose methylation of the first transcribed nucleotide are required for hMTr2, but the presence of cap1 methylation increases hMTr2 activity. The hMTr2 protein is distributed throughout the nucleus and cytosol, in contrast to the nuclear hMTr1. The details of how and why specific transcripts undergo modification with these ribose methylations remains to be elucidated. The 2′-O-ribose RNA cap methyltransferases are present in varying combinations in most eukaryotic and many viral genomes. With the capping enzymes in hand their biological purpose can be ascertained.
Medial compartment osteoarthritis is a common disorder that often is treated by unicompartmental knee arthroplasty (UKA). Although the Oxford 3 prosthesis is commonly used based on revision rate and cumulative survival, our experience suggests that although there may be adequate implant survival rates, we observed a worrisome and undisclosed reintervention rate of nonrevision procedures.
We describe the frequency and cause of repeat intervention subsequent to implanting this device.
Between 1998 and 2005, 398 patients underwent UKA using the Oxford 3 prosthesis. The minimum followup was 12 months (mean, 43 months; range, 12–102 months).
Forty of the 398 (10%) patients had 55 (13.8%) repeat anesthetics (reintervention). There were 38 nonrevision reinterventions. Revision was performed in 15 patients (3.8%), but two patients had a second revision (17 revisions or 4.3%). We revised the UKA to a second UKA in seven of the 15 cases but two subsequently were rerevised to a TKA; eight were revised directly to a TKA.
Although our data confirm the reported revision rates for this prosthesis, we observed a substantial reintervention rate. Most of the reinterventions are minor and are diagnosed frequently and treated arthroscopically. If revision is required, a second UKA may be considered and performed successfully in patients with isolated loosening of one component.
Level of Evidence
Level II, prognostic study. See Guidelines for Authors for a complete description of levels of evidence.
Compounds that inhibit signalling upstream of ERK (extracellular-signal-regulated kinase) are promising anticancer therapies, motivating research to define how this pathway promotes cancers. In the present study, we show that human capicúa represses mRNA expression for PEA3 (polyoma enhancer activator 3) Ets transcription factors ETV1, ETV4 and ETV5 (ETV is Ets translocation variant), and this repression is relieved by multisite controls of capicúa by ERK, p90RSK (p90 ribosomal S6 kinase) and 14-3-3 proteins. Specifically, 14-3-3 binds to p90RSK-phosphorylated Ser173 of capicúa thereby modulating DNA binding to its HMG (high-mobility group) box, whereas ERK phosphorylations prevent binding of a C-terminal NLS (nuclear localization sequence) to importin α4 (KPNA3). ETV1, ETV4 and ETV5 mRNA levels in melanoma cells are elevated by siRNA (small interfering RNA) knockdown of capicúa, and decreased by inhibiting ERK and/or expressing a form of capicúa that cannot bind to 14-3-3 proteins. Capicúa knockdown also enhances cell migration. The findings of the present study give further mechanistic insights into why ETV1 is highly expressed in certain cancers, indicate that loss of capicúa can desensitize cells to the effects of ERK pathway inhibitors, and highlight interconnections among growth factor signalling, spinocerebellar ataxias and cancers.
cancer; capicúa; Ets translocation variant 1 (ETV1); 14-3-3 protein; spinocerebellar ataxia type 1 (SCA1); B2M, β2 microglobuluin; CRE, CIC-responsive element; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; DUX4, Double homeobox 4; ECL, enhanced chemiluminescence; EGF, epidermal growth factor; EMSA, electrophoretic mobility-shift assay; ERK, extracellular-signal-regulated kinase; ETV, Ets translocation variant; EWS, Ewing sarcoma protein; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GIST, gastrointestinal stromal tumour; HA, haemagglutinin; HEK, human embryonic kidney; HMG, high-mobility group; IGF1, insulin-like growth factor 1; KPNA3, importin α4/karyopherin α3; LC, liquid chromatography; MS/MS, tandem MS; NLS, nuclear localization sequence; p90RSK, p90 ribosomal S6 kinase; PEA3, polyoma enhancer activator 3; PDK1, phosphoinositide-dependent kinase 1; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; PKC, protein kinase C; RT, reverse transcription; SCA, spinocerebellar ataxia; siRNA, small interfering RNA
This report from a single institution describes the results of consecutive pediatric heart operations done by trainees under the supervision of a senior surgeon. The 3.1% mortality seen in 1067 index operations is comparable across procedures and risk bands to risk-stratified results reported by the Society of Thoracic Surgeons. With appropriate mentorship, surgeons-in-training are able to achieve good results as first operators.
LRRK2 (leucine-rich repeat protein kinase 2) is mutated in a significant number of Parkinson's disease patients, but still little is understood about how it is regulated or functions. In the present study we have demonstrated that 14-3-3 protein isoforms interact with LRRK2. Consistent with this, endogenous LRRK2 isolated from Swiss 3T3 cells or various mouse tissues is associated with endogenous 14-3-3 isoforms. We have established that 14-3-3 binding is mediated by phosphorylation of LRRK2 at two conserved residues (Ser910 and Ser935) located before the leucine-rich repeat domain. Our results suggests that mutation of Ser910 and/or Ser935 to disrupt 14-3-3 binding does not affect intrinsic protein kinase activity, but induces LRRK2 to accumulate within discrete cytoplasmic pools, perhaps resembling inclusion bodies. To investigate links between 14-3-3 binding and Parkinson's disease, we studied how 41 reported mutations of LRRK2 affected 14-3-3 binding and cellular localization. Strikingly, we found that five of the six most common pathogenic mutations (R1441C, R1441G, R1441H, Y1699C and I2020T) display markedly reduced phosphorylation of Ser910/Ser935 thereby disrupting interaction with 14-3-3. We have also demonstrated that Ser910/Ser935 phosphorylation and 14-3-3 binding to endogenous LRRK2 is significantly reduced in tissues of homozygous LRRK2(R1441C) knock-in mice. Consistent with 14-3-3 regulating localization, all of the common pathogenic mutations displaying reduced 14-3-3-binding accumulated within inclusion bodies. We also found that three of the 41 LRRK2 mutations analysed displayed elevated protein kinase activity (R1728H, ~2-fold; G2019S, ~3-fold; and T2031S, ~4-fold). These results provide the first evidence suggesting that 14-3-3 regulates LRRK2 and that disruption of the interaction of LRRK2 with 14-3-3 may be linked to Parkinson's disease.
cytoplasmic localization; 14-3-3 protein; leucine-rich repeat protein kinase 2 (LRRK2); Parkinson's disease; pathogenic mutation; phosphorylation; CDC, cell division cycle; DIG, digoxigenin; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; FBS, fetal bovine serum; GFP, green fluorescent protein; HEK-293, human embryonic kidney; Hsp90, heat-shock protein 90; IPI, International Protein Index; KLH, keyhole-limpet haemocyanin; LRRK2, leucine-rich repeat protein kinase 2; MARK3, microtubule affinity-regulating kinase 3; PD, Parkinson's disease; ROC, Ras of complex GTPase domain; COR, C-terminal of ROC; SILAC, stable isotope labelling of amino acids; TBST, Tris-buffered saline with Tween 20
Lycopene, the red pigment of tomatoes, is a carotenoid with potent antioxidant properties. Although lycopene might function as a prostate cancer chemoprevention agent, little is known about its effects at the cellular level. To define general changes induced by treatment of cells with lycopene, and to gain insights into the possible chemoprevention properties of lycopene, we investigated changes in protein expression after lycopene treatment in human LNCaP cells. The high throughput proteomics data were then visualized and analyzed by novel biological protein pathway modeling software. Differentially expressed proteins were identified, and the data were analyzed by protein pathway simulation software without need for specialized programming by importing pathway models from a number of sources or creating their own. One notable outcome was the identification of a group of upregulated proteins involved in detoxification of reactive oxygen species. This finding suggests that a possible mechanism of lycopene chemoprevention is the stimulation of detoxification enzymes associated with the antioxidant response element. Novel biological pathway modeling software enhances analysis of large proteomics data. When applied to the analysis of proteins differentially expressed in prostate cancer cells upon treatment with lycopene, the upregulation of detoxification enzymes was identified.
detoxification enzymes; ICAT; LNCaP; lycopene; teranode
Spliced leader (SL) trans-splicing is a common mRNA processing mechanism in dinoflagellates, in which a 22-nt sequence is transferred from the 5′-end of a small noncoding RNA, the SL RNA, to the 5′-end of mRNA molecules. Although the SL RNA gene was shown initially to be organized as tandem repeats with transcripts of 50–60 nt, shorter than most of their counterparts in other organisms, other gene organizations and transcript lengths were reported subsequently. To address the evolutionary gradient of gene organization complexity, we thoroughly examined transcript and gene organization of the SL RNA in a phylogenetically and ecologically diverse group of dinoflagellates representing four Orders. All these dinoflagellates possessed SL RNA transcripts of 50–60 nt, although in one species additional transcripts of up to 92 nt were also detected. At the genomic level, various combinations of SL RNA and 5S rRNA tandem gene arrays, including SL RNA–only, 5S rRNA–only, and mixed SL RNA–5S rRNA (SL–5S) clusters, were amplified by polymerase chain reaction for six dinoflagellates, containing intergenic spacers ranging from 88 bp to over 1.2 kb. Of these species, no SL–5S cluster was detected in Prorocentrum minimum, and only Karenia brevis showed the U6 small nuclear RNA gene associated with these mixed arrays. The 5S rRNA–only array was also found in three dinoflagellates, along with two SL–5S-adjacent arrangements found in two other species that could represent junctions. Two species contained multimeric SL exon repeats with no associated intron. These results suggest that 1) both the SL RNA tandem repeat and the SL–5S cluster genomic organizations are an “ancient” and widespread feature within the phylum of dinoflagellates and 2) rampant genomic duplication and recombination are ongoing independently in each dinoflagellate lineage, giving rise to the highly complex and diversified genomic arrangements of the SL RNA gene, while conserving the length and structure of the functional SL RNA.
Dinoflagellate; SL RNA; complex genomic arrangement
Although the kidney is a major organ for vitamin D metabolism, activity, and calcium-related homeostasis, little is known about whether this nutrient plays a role in the development or the inhibition of kidney cancer. To address this gap in knowledge, the authors examined the association between circulating 25-hydroxyvitamin D (25(OH)D) and kidney cancer within a large, nested case-control study developed as part of the Cohort Consortium Vitamin D Pooling Project of Rarer Cancers. Concentrations of 25(OH)D were measured from 775 kidney cancer cases and 775 age-, sex-, race-, and season-matched controls from 8 prospective cohort studies. Overall, neither low nor high concentrations of circulating 25(OH)D were significantly associated with kidney cancer risk. Although the data showed a statistically significant decreased risk for females (odds ratio = 0.31, 95% confidence interval: 0.12, 0.85) with 25(OH)D concentrations of ≥75 nmol/L, the linear trend was not statistically significant and the number of cases in this category was small (n = 14). The findings from this consortium-based study do not support the hypothesis that vitamin D is inversely associated with the risk of kidney cancer overall or with renal cell carcinoma specifically.
case-control studies; cohort studies; kidney neoplasms; prospective studies; vitamin D
Upper gastrointestinal (GI) cancers of the stomach and esophagus have high incidence and mortality worldwide, but they are uncommon in Western countries. Little information exists on the association between vitamin D and risk of upper GI cancers. This study examined the association between circulating 25-hydroxyvitamin D (25(OH)D) and upper GI cancer risk in the Cohort Consortium Vitamin D Pooling Project of Rarer Cancers. Concentrations of 25(OH)D were measured from 1,065 upper GI cancer cases and 1,066 age-, sex-, race-, and season-of blood draw–matched controls from 8 prospective cohort studies. In multivariate-adjusted models, circulating 25(OH)D concentration was not significantly associated with upper GI cancer risk. Subgroup analysis by race showed that among Asians, but not Caucasians, lower concentrations of 25(OH)D (<25 nmol/L) were associated with a statistically significant decreased risk of upper GI cancer (reference: 50–<75 nmol/L) (odds ratio = 0.53, 95% confidence interval: 0.31, 0.91; P trend = 0.003). Never smokers with concentrations of <25 nmol/L showed a lower risk of upper GI cancers (odds ratio = 0.55, 95% confidence interval: 0.31, 0.96). Subgroup analyses by alcohol consumption produced opposing trends. Results do not support the hypothesis that interventions aimed at increasing vitamin D status would lead to a lower risk of these highly fatal cancers.
case-control studies; cohort studies; esophageal neoplasms; prospective studies; stomach neoplasms; vitamin D
Through trans-splicing of a 39-nt Spliced Leader (SL) onto each protein-coding transcript, mature kinetoplastid mRNA acquire a hypermethylated 5′-cap structure, but its function has been unclear. Gene deletions for three Trypanosoma brucei cap 2′-O-ribose methyltransferases, TbMTr1, TbMTr2, and TbMTr3, reveal distinct roles for four 2′-O-methylated nucleotides. Elimination of individual gene pairs yields viable cells, however attempts at double knockouts resulted in the generation of a TbMTr2−/−/TbMTr3−/− cell line only. Absence of both kinetoplastid-specific enzymes in TbMTr2−/−/TbMTr3−/− lines yielded substrate SL RNA and mRNA with cap 1. TbMTr1−/− translation is comparable to wildtype, while cap 3 and cap 4 loss reduced translation rates, exacerbated by the additional loss of cap 2. TbMTr1−/− and TbMTr2−/−/TbMTr3−/− lines grow to lower densities under normal culture conditions relative to wildtype cells, with growth rate differences apparent under low serum conditions. Cell viability may not tolerate delays at both the nucleolar Sm-independent and nucleoplasmic Sm-dependent stages of SL RNA maturation combined with reduced rates of translation. A minimal level of mRNA cap ribose methylation is essential for trypanosome viability, providing the first functional role for the cap 4.
gene knockout; methyltransferase; ribose 2′-O-methylation; SL RNA; spliced leader; trans-splicing
More than 200 phosphorylated 14-3-3-binding sites in the literature were analysed to define 14-3-3 specificities, identify relevant protein kinases, and give insights into how cellular 14-3-3/phosphoprotein networks work. Mode I RXX(pS/pT)XP motifs dominate, although the +2 proline residue occurs in less than half, and LX(R/K)SX(pS/pT)XP is prominent in plant 14-3-3-binding sites. Proline at +1 is rarely reported, and such motifs did not stand up to experimental reanalysis of human Ndel1. Instead, we discovered that 14-3-3 interacts with two residues that are phosphorylated by basophilic kinases and located in the DISC1 (disrupted-in-schizophrenia 1)-interacting region of Ndel1 that is implicated in cognitive disorders. These data conform with the general findings that there are different subtypes of 14-3-3-binding sites that overlap with the specificities of different basophilic AGC (protein kinase A/protein kinase G/protein kinase C family) and CaMK (Ca2+/calmodulin-dependent protein kinase) protein kinases, and a 14-3-3 dimer often engages with two tandem phosphorylated sites, which is a configuration with special signalling, mechanical and evolutionary properties. Thus 14-3-3 dimers can be digital logic gates that integrate more than one input to generate an action, and coincidence detectors when the two binding sites are phosphorylated by different protein kinases. Paired sites are generally located within disordered regions and/or straddle either side of functional domains, indicating how 14-3-3 dimers modulate the conformations and/or interactions of their targets. Finally, 14-3-3 proteins bind to members of several multi-protein families. Two 14-3-3-binding sites are conserved across the class IIa histone deacetylases, whereas other protein families display differential regulation by 14-3-3s. We speculate that 14-3-3 dimers may have contributed to the evolution of such families, tailoring regulatory inputs to different physiological demands.
14-3-3 protein; AGC protein kinase; Ca2+/calmodulin-dependent protein kinase; disrupted-in-schizophrenia 1 (DISC1); evolution; AANAT, serotonin acetyltransferase; AGC, protein kinase A/protein kinase G/protein kinase C family kinase; AMPK, AMP-activated protein kinase; BAD, Bcl-XL/Bcl-2-associated death promoter; CaMK, Ca2+/calmodulin-dependent protein kinase; CDK5, cyclin-dependent kinase 5; DIG, digoxigenin; DISC1, disrupted-in-schizophrenia 1; DSTT, Division of Signal Transduction Therapy; EST, expressed sequence tag; FOXO, Forkhead box O; GLUT4, glucose transporter 4; GST, glutathione transferase; HA, haemagglutinin; HAP1A, Huntingtin-associated protein 1A; HDAC, histone deacetylase; HEK, human embryonic kidney; KLC, kinesin light chain; MARK, microtubule affinity-regulating kinase; PI4K, phosphoinositide 4-kinase; PKB, protein kinase B; PKC, protein kinase C; PP2A, protein phosphatase 2A; RSK, ribosomal S6 kinase; YAP1, yes-associated protein 1
A proof-of-concept demonstration of the use of label-free quantitative glycoproteomics for biomarker discovery workflow is presented here, using a mouse model for skin cancer as an example. Blood plasma was collected from 10 control mice, and 10 mice having a mutation in the p19ARF gene, conferring them high propensity to develop skin cancer after carcinogen exposure. We enriched for N-glycosylated plasma proteins, ultimately generating deglycosylated forms of the modified tryptic peptides for liquid chromatography mass spectrometry (LC-MS) analyses. LC-MS runs for each sample were then performed with a view to identifying proteins that were differentially abundant between the two mouse populations. We then used a recently developed computational framework, Corra, to perform peak picking and alignment, and to compute the statistical significance of any observed changes in individual peptide abundances. Once determined, the most discriminating peptide features were then fragmented and identified by tandem mass spectrometry with the use of inclusion lists. We next assessed the identified proteins to see if there were sets of proteins indicative of specific biological processes that correlate with the presence of disease, and specifically cancer, according to their functional annotations. As expected for such sick animals, many of the proteins identified were related to host immune response. However, a significant number of proteins also directly associated with processes linked to cancer development, including proteins related to the cell cycle, localisation, trasport, and cell death. Additional analysis of the same samples in profiling mode, and in triplicate, confirmed that replicate MS analysis of the same plasma sample generated less variation than that observed between plasma samples from different individuals, demonstrating that the reproducibility of the LC-MS platform was sufficient for this application. These results thus show that an LC-MS-based workflow can be a useful tool for the generation of candidate proteins of interest as part of a disease biomarker discovery effort.
Skin cancer; LC-MS; Label-free protein quantification; Biomarker discovery; Systems biology; Targeted peptide sequencing; Glycoproteomics; Plasma
Kinetoplastid flagellates attach a 39-nucleotide spliced leader (SL) upstream of protein-coding regions in polycistronic RNA precursors through trans splicing. SL modifications include cap 2′-O-ribose methylation of the first four nucleotides and pseudouridine (ψ) formation at uracil 28. In Trypanosoma brucei, TbMTr1 performs 2′-O-ribose methylation of the first transcribed nucleotide, or cap 1. We report the characterization of an SL RNA processing complex with TbMTr1 and the SLA1 H/ACA small nucleolar ribonucleoprotein (snoRNP) particle that guides SL ψ28 formation. TbMTr1 is in a high-molecular-weight complex containing the four conserved core proteins of H/ACA snoRNPs, a kinetoplastid-specific protein designated methyltransferase-associated protein (TbMTAP), and the SLA1 snoRNA. TbMTAP-null lines are viable but have decreased SL RNA processing efficiency in cap methylation, 3′-end maturation, and ψ28 formation. TbMTAP is required for association between TbMTr1 and the SLA1 snoRNP but does not affect U1 small nuclear RNA methylation. A complex methylation profile in the mRNA population of TbMTAP-null lines indicates an additional effect on cap 4 methylations. The TbMTr1 complex specializes the SLA1 H/ACA snoRNP for efficient processing of multiple modifications on the SL RNA substrate.
Many components of the RNA polymerase II transcription machinery have been identified in kinetoplastid protozoa, but they diverge substantially from other eukaryotes. Furthermore, protein-coding genes in these organisms lack individual transcriptional regulation, since they are transcribed as long polycistronic units. The transcription initiation sites are assumed to lie within the 'divergent strand-switch' regions at the junction between opposing polycistronic gene clusters. However, the mechanism by which Kinetoplastidae initiate transcription is unclear, and promoter sequences are undefined.
The chromosomal location of TATA-binding protein (TBP or TRF4), Small Nuclear Activating Protein complex (SNAP50), and H3 histones were assessed in Leishmania major using microarrays hybridized with DNA obtained through chromatin immunoprecipitation (ChIP-chip). The TBP and SNAP50 binding patterns were almost identical and high intensity peaks were associated with tRNAs and snRNAs. Only 184 peaks of acetylated H3 histone were found in the entire genome, with substantially higher intensity in rapidly-dividing cells than stationary-phase. The majority of the acetylated H3 peaks were found at divergent strand-switch regions, but some occurred at chromosome ends and within polycistronic gene clusters. Almost all these peaks were associated with lower intensity peaks of TBP/SNAP50 binding a few kilobases upstream, evidence that they represent transcription initiation sites.
The first genome-wide maps of DNA-binding protein occupancy in a kinetoplastid organism suggest that H3 histones at the origins of polycistronic transcription of protein-coding genes are acetylated. Global regulation of transcription initiation may be achieved by modifying the acetylation state of these origins.
A monitoring campaign was conducted in August-September 2005 to compare different experimental approaches quantifying school bus self-pollution. As part of this monitoring campaign, a detailed characterization of PM2.5 diesel engine emissions from the tailpipe and crankcase emissions from the road draft tubes was performed. To distinguish between tailpipe and crankcase vent emissions, a deuterated alkane, n-hexatriacontane-d74 (n-C36D74) was added to the engine oil to serve as intentional quantitative tracers for lubricating oil PM emissions. This paper focuses on the detailed chemical speciation of crankcase and tailpipe PM emissions from two school buses used in this study. We found that organic carbon emission rates were generally higher from the crankcase than from the tailpipe for these two school buses, while elemental carbon contributed significantly only in the tailpipe emissions. The n-C36D74 that was added to the engine oil was emitted at higher rates from the crankcase than the tailpipe. Tracers of engine oil (hopanes, and steranes) were present in much higher proportion in crankcase emissions. Particle-associated PAH emission rates were generally very low (< 1 μg/km), but more PAH species were present in crankcase than in tailpipe emissions. The speciation of samples collected in the bus cabins was consistent with most of the bus self-pollution originating from crankcase emissions.
School bus self-pollution; tailpipe and crankcase emissions; organic speciation; tracers