[Identification of proteins associated with transcription factors HOXA9 and E2A-PBX1 by tandem affinity purification].

Chimeric transcription factor E2A-PBX1 induces the development of acute lymphoblastic B-cell leukemia in children. Using a transgenic mouse model, we previously demonstrated that homeobox (HOX) gene HOXA9 genetically interact with E2A-PBX1 gene in the development of B-cell leukemia in mice. HOXA9 itself is a potent oncogene resulting in myeloid leukemia when overexpressed, which is strongly accelerated by its collaborator Meis1. HOX, PBX1 and MEIS1 proteins have been shown to form hetero dimeric or trimeric complexes in different combinations. Cooperative interaction between PBX1 and HOX proteins enhances their DNA binding specificity, essential for HOX dependent developmental programs. PBX1 is retained in E2A-PBX1, and thus the strong transcriptional activator properties of E2A-PBX1 may lead to aberrant activation of normally repressed targets of HOX-PBX complexes. However, although there is evidence that E2A-PBX1 could bind to HOX and MEIS1 proteins it is still unclear whether such complexes are actually required for leukemic transformation or whether E2A-PBX1 and HOXA9 are each part of larger protein complexes acting in independent complementing oncogenic pathways. In this study we aim to search for other HOXA9 and E2A-PBX1 interacting proteins. To identify novel proteins interacting with human E2A-PBX1 or HOXA9 we used tandem affinity purification (TAP) of protein complexes from 697 pre-B leukemic and HeLa cell lines transduced to express E2A-PBX1 or HOXA9, respectively, with covalently attached FLAG/HA peptides. The protein composition of each complex was determined using tandem mass-spectrometry. In the E2A-PBX1 containing complex we identified lymphoid transcription factor IKAROS, chromatin remodeling factors of SWI/SNF family while multiple subunits of translation initiation factor eIF3, E3 ubiquitin ligase UBR5 emerged from the HOXA9 complex as potential critical protein partners. This is the first time the protein partners of either E2A-PBX1 or HOXA9 oncoproteins were identified using an unbiased biochemical approach. The identification of translation initiation factors associated with HOXA9 might indicate a novel function for HOX proteins independent of their transcriptional activity.

SUMOylation- and GAR1-Dependent Regulation of Dyskerin Nuclear and Subnuclear Localization.

The nuclear and subnuclear compartmentalization of the telomerase-associated protein and H/ACA ribonucleoprotein component dyskerin is an important although incompletely understood aspect of H/ACA ribonucleoprotein function. Four SUMOylation sites were previously identified in the C-terminal nuclear/nucleolar localization signal (N/NoLS) of dyskerin. We found that a cytoplasmic localized C-terminal truncation variant of dyskerin lacking most of the C-terminal N/NoLS represents an under-SUMOylated variant of dyskerin compared to wild-type dyskerin. We demonstrate that mimicking constitutive SUMOylation of dyskerin using a SUMO3 fusion construct can drive nuclear accumulation of this variant and that the SUMO site K467 in this N/NoLS is particularly important for the subnuclear localization of dyskerin to the nucleolus in a mature H/ACA complex assembly- and SUMO-dependent manner. We also characterize a novel SUMO-interacting motif in the mature H/ACA complex component GAR1 that mediates the interaction between dyskerin and GAR1. Mislocalization of dyskerin, either in the cytoplasm or excluded from the nucleolus, disrupts dyskerin function and leads to reduced interaction of dyskerin with the telomerase RNA. These data indicate a role for dyskerin C-terminal N/NoLS SUMOylation in regulating the nuclear and subnuclear localization of dyskerin, which is essential for dyskerin function as both a telomerase-associated protein and as an H/ACA ribonucleoprotein.

Proteogenomic-based discovery of minor histocompatibility antigens with suitable features for immunotherapy of hematologic cancers.

Pre-clinical studies have shown that injection of allogeneic T cells primed against a single minor histocompatibility antigen (MiHA) could cure hematologic cancers (HC) without causing any toxicity to the host. However, translation of this approach in humans has been hampered by the paucity of molecularly defined human MiHAs. Using a novel proteogenomic approach, we have analyzed cells from 13 volunteers and discovered a vast repertoire of MiHAs presented by the most common HLA haplotype in European Americans: HLA-A*02:01;B*44:03. Notably, out of >6000 MiHAs, we have identified a set of 39 MiHAs that share optimal features for immunotherapy of HCs. These 'optimal MiHAs' are coded by common alleles of genes that are preferentially expressed in hematopoietic cells. Bioinformatic modeling based on MiHA allelic frequencies showed that the 39 optimal MiHAs would enable MiHA-targeted immunotherapy of practically all HLA-A*02:01;B*44:03 patients. Further extension of this strategy to a few additional HLA haplotypes would allow treatment of almost all patients.

Acyclic oligonucleotide analogues.

Acyclic analogues of oligothymidylate and oligoadenylate and their alternating copolymers were synthesized to study their thermal melting, their stability against snake venom phosphodiesterase and their primer/template properties using the Klenow fragment of the Escherichia coli DNA polymerase I enzyme. Acyclic dodecaadenylate (GlyA)12 hybridized to dodecathymidylate p(dT)12, and the complex presented a sharp melting with a Tm at 24 degrees C. This association was confirmed by circular dichroism curves which were similar to those of the natural oligonucleotide duplexes in A-conformation. (GlyA)12 proved very stable against snake venom phosphodiesterase hydrolysis. The reaction rate was more than 10,000 times slower than that of p(dT)12. (GlyA)12 served as a primer for the Klenow DNA polymerase. When (GlyA)12 was complexed with the poly(dT) template, the enzyme polymerized dATP but the reaction was much slower than with the (GlyT)12 primer. Molecular modelling of atactic (GlyA)12.(dT)12 of the A-conformation indicates that this conformation is energetically possible.

Characterization of a solid-phase extraction device for discontinuous on-line preconcentration in capillary electrophoresis-based peptide mapping.

Peptide mapping by capillary electrophoresis (CE) with UV detection is problematic for the characterization of proteins that can only be obtained at low micromolar concentrations. Dilution of peptide fragments during digestion of the protein can further reduce the detection sensitivity in peptide mapping to the point where analysis at sub-micromolar concentrations is not possible. A remedy to this problem is preconcentration (sample enrichment) of the proteolytic digest by solid-phase extraction (SPE). To minimize non-specific adsorptive losses during sample handling, on-line SPE-CE is preferred. However, packed-inlet SPE-CE is not always feasible due to either instrument or sample limitations. We describe here a simple method of preconcentration by discontinuous on-line SPE-CE, specifically applied to peptide mapping in low-pH separation buffer after protein digestion in a solid-phase enzyme microreactor. The SPE-CE system does not require application of a low pressure during electrophoretic separation to overcome reversed electroosmotic flow because the preconcentrator device is disconnected from the separation capillary before the electric field is applied. Up to a 500-fold preconcentration factor can be achieved with this device, which can be reused for many samples. Parameters such as the volume of desorption solution, the adsorption/desorption (chromatographic) process, reproducibility of packing the SPE preconcentrator and effects of sample concentration on the peptide map are investigated.

On-line solid-phase preconcentration for sensitivity enhancement in capillary electrophoresis.

Capillary electrophoresis is attractive for the analysis of biological samples because only a few nanoliters of sample need to be injected. Indeed, optimal resolution is achieved when the injected volume is 1% or less of the total capillary volume. Unfortunately, this advantage leads to severe detection limitations compounded by the fact that many analytes in a biological sample are present at very low concentrations. To overcome the detection sensitivity limitations of CE, nonspecific, on-line preconcentration has been employed in a variety of applications. This technique is based on inserting a small quantity of reversed-phase material (e.g., C18 particles or membrane) near the inlet of the CE capillary for sample enrichment by solid-phase extraction. A detachable cartridge containing the solid phase is relatively simple to construct and permits the injection of large sample volumes (1-100 microL) into the capillary. Elution of adsorbed analyte in less than 100 nL of solvent permits a 100-1000 fold improvement in the effective concentration limit of detection, depending on the hydrophobicity of the compound. Detection of analytes present in complex mixtures at concentrations as low as 20 amol/microL has been reported when using an on-line preconcentration device in conjunction with CE-mass spectrometry. In this review, the potential merits of the technique are described and some examples of direct analysis of biological samples without rigorous off-line pretreatment are given.

On-line system for peptide mapping by capillary electrophoresis at sub-micromolar concentrations.

Peptide mapping has been widely used for the identification of modified proteins involved in certain diseases. Despite the fact that capillary electrophoresis (CE) has been shown to be a powerful tool for the separation and detection of tryptic peptide fragments after protein digestion, this technique lacks sensitivity for mapping proteins isolated in very small quantities from biological samples. Consequently, it has been necessary to preconcentrate the protein before adding the proteolytic enzyme for digestion in solution. These experimental steps are quite long, labor intensive and require a lot of sample handling. In this paper, we describe an on-line system allowing digestion of the protein, followed by preconcentration, separation and detection of the tryptic fragments in 4 h. Up to an 800-fold preconcentration factor was achieved for cytochrome c, despite a loss of separation efficiency induced by the multiple-valve design of the system and dispersion of the 60-nl desorption plug. Moreover, our system showed good migration time reproducibility between peptide maps and could be reused for several samples.