Spare PRELI Gene Loci: Failsafe Chromosome Insurance?

LEA (late embryogenesis abundant) proteins encode conserved N-terminal mitochondrial signal domains and C-terminal (A/TAEKAK) motif repeats, long-presumed to confer cell resistance to stress and death cues. This prompted the hypothesis that LEA proteins are central to mitochondria mechanisms that connect bioenergetics with cell responses to stress and death signaling. In support of this hypothesis, recent studies have demonstrated that mammalian LEA protein PRELI can act as a biochemical hub, which upholds mitochondria energy metabolism, while concomitantly promoting B cell resistance to stress and induced death. Hence, it is important to define in vivo the physiological relevance of PRELI expression.Given the ubiquitous PRELI expression during mouse development, embryo lethality could be anticipated. Thus, conditional gene targeting was engineered by insertion of flanking loxP (flox)/Cre recognition sites on PRELI chromosome 13 (Chr 13) locus to abort its expression in a tissue-specific manner. After obtaining mouse lines with homozygous PRELI floxed alleles (PRELI(f/f)), the animals were crossed with CD19-driven Cre-recombinase transgenic mice to investigate whether PRELI inactivation could affect B-lymphocyte physiology and survival. Mice with homozygous B cell-specific PRELI deletion (CD19-Cre/Chr13 PRELI(-/-)) bred normally and did not show any signs of morbidity. Histopathology and flow cytometry analyses revealed that cell lineage identity, morphology, and viability were indistinguishable between wild type CD19-Cre/Chr13 PRELI(+/+) and CD19-Cre/Chr13 PRELI(-/-) deficient mice. Furthermore, B cell PRELI gene expression seemed unaffected by Chr13 PRELI gene targeting. However, identification of additional PRELI loci in mouse Chr1 and Chr5 provided an explanation for the paradox between LEA-dependent cytoprotection and the seemingly futile consequences of Chr 13 PRELI gene inactivation. Importantly, PRELI expression from spare gene loci appeared ample to surmount Chr 13 PRELI gene deficiency.These findings suggest that PRELI is a vital LEA B cell protein with failsafe genetics

<i>PRELI</i> motifs and their relevance in subcellular location and function.

<p>(<b>a</b>) Hypothetical <i>PRELI</i> structure based on queries of deduced amino acid sequence against protein databases (<a href="http://wwwuniprot.org/uniprot/Q8R107" target="_blank">http://wwwuniprot.org/uniprot/Q8R107</a>; <a href="http://pfam.sanger.ac.uk/family/PF04707" target="_blank">http://pfam.sanger.ac.uk/family/PF04707</a>). <i>PRELI</i> MSF1-like region is depicted as a dark grey octagonal form, while its <i>LEA</i> motif is shown as theoretical α helices, flanked by low complexity (LC) domains (lighter grey rectangles). (<b>b</b>) Confocal microscopy results on control (Vector) and <i>PRELI</i> PBlin-1 transfectants <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037949#pone.0037949-McKeller1" target="_blank">[5]</a> confirm MSF1-like deduced prediction that N-terminal signal peptide <b>mvkyflgqsvlrsswdqvfaafwqrypnpyskhvl</b> can direct <i>PRELI</i> expression (red fluorescence) into the mitochondria, as shown by its co-localization (merge) with mitochondrial HSP60 (green fluorescence) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037949#pone.0037949-McKeller1" target="_blank">[5]</a>. (<b>c</b>) Confocal microscopy results show that in contrast to control cells (Control), <i>PRELI</i> Blin-1 transfectants (<i>PRELI</i>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037949#pone.0037949-McKeller1" target="_blank">[5]</a> prevent STS-induced (+STS) release of <i>AIF</i> from the mitochondria and uphold cell morphology and survival. These results are consistent with the conserved cytoprotection function of <i>LEA</i>-containing proteins. <i>AIF</i> is shown in red fluorescence within greyscale cell background.</p

Spare <i>PRELI</i> gene copies in humans.

<p>This figure provides mRNA and protein database URL information, including gene ID annotations, chromosome coordinates, nucleotide and protein accession number annotations for human PRELI gene duplicates found in Chr 5q35.3 and Chr 6q22.32.</p

<i>PRELI</i> transcription by CD19Cre/Ch13 <i>PRELI</i> mice.

<p>(<b>a</b>) Northern blots compare PRELI mRNA levels between WT CD19/Cre/Chr13 <i>PRELI</i>^+/+ and heterozygous CD19-Cre/Chr13 <i>PRELI</i>^+/− or homozygous CD19-Cre/Chr13 <i>PRELI</i>^−/− mouse spleen B cells. (<b>b</b>) qRT-PCR assessment of <i>PRELI</i> transcription between WT CD19/Cre/Chr13 <i>PRELI</i>^+/+ and homozygousCD19-Cre/Chr13 <i>PRELI</i>^−/− mice. qRT-PCR comparisons include results obtained with RNA from bone marrow and spleen B cells. Data are presented in relative Ct and are the result of at least three WT CD19/Cre/Chr13 <i>PRELI</i>^+/+ and CD19-Cre/Chr13 <i>PRELI</i>^−/− paired littermates.</p

CD19-Cre/Ch13 <i>PRELI</i>^−/− transcripts have exon II sequences.

<p>(<b>a</b>) RT-PCR results of peripheral blood leukocyte (PBL) <i>PRELI</i> transcript amplification from WT CD19/Cre/Chr13 <i>PRELI</i>^+/+, heterozygous CD19-Cre/Chr13 <i>PRELI</i>^+/−, and homozygous CD19-Cre/Chr13 <i>PRELI</i>^−/− mouse littermates. A 2.5–0.3 kb molecular weight marker range is shown on the left of the ethidium bromide-stained gel image to verify the length of <i>PRELI</i> open reading frame (ORF). The predicted 670 bp molecular size of <i>PRELI</i> ORF PCR fragments includes forward and reverse amplifying primers with respective restriction endonuclease EcoR1 and Sal1 sequences (depicted in section c). (<b>b</b>) Immunoblot results reveal PBL PRELI protein levels in WT CD19/Cre/Chr13 <i>PRELI</i>^+/+, heterozygous CD19-Cre/Chr13 <i>PRELI</i>^+/−, and homozygous CD19-Cre/Chr13 <i>PRELI</i>^−/− mice. Of note, the same blot was divided into high and low molecular mass (kDa) sections, indicated by the intersect mark (≠) at proportional kDa boundaries. This strategy enables the assessment of PRELI expression (24.9 kDa) and at the same time, permits to independently monitor protein loading (MSF1-like) on identical sample lanes. (<b>c</b>) cDNA sequencing results obtained from a representative homozygous CD19-Cre/Chr13 PRELI^−/− clone obtained after RT-PCR amplification (section a). Amplifying forward and reverse primers are shown in bold font and respective EcoR1 and Sal1 restriction endonuclease target sequences are indicated in grey font. Canonical translation initiation and stop codon sequences are circled, while exon II is framed. This sequence is identical to NCBI database annotation XM_001476721.</p

Cell lineage phenotype analyses of CD19Cre/Chr13 <i>PRELI</i> mice.

<p>Flow cytometry results compare the presence of B220⁺ (left histograms) and CD19⁺ (right histograms) lymphocytes in peripheral blood leukocytes of WT CD19/Cre/Chr13 <i>PRELI</i>^+/+ (top), heterozygous CD19-Cre/Chr13 <i>PRELI</i>^+/− (middle), and homozygous CD19-Cre/Chr13 <i>PRELI</i>^−/− mice (bottom). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037949#s2" target="_blank">Results</a> are presented as number of cells (%) with relevant fluorescence (Log) staining. The figure is a representative result of at least three independent analyses performed with age-matched littermates (n  =  3). Standard error mean (± SEM) bars are indicated and high statistical significance values scored p≤0.001.</p

<i>PRELI</i> mRNA expression during mouse embryo development and in adult life.

<p>(<b>a</b>) Northern blot results show the prominent <i>PRELI</i> mRNA expression at 7–17 days post coitum (dpc) stages of mouse embryo development. (<b>b</b>) <i>In situ</i> hybridization results of whole-mount embryo sections show robust and ubiquitous <i>PRELI</i> mRNA expression at 13-dpc developmental stage. Left: hybridization with digoxigenin-labeled anti-sense <i>PRELI</i> complementary RNA (cRNA) probe shows prominent red fluorescence overlapping with Hoechst blue fluorescence counterstain. Right: hybridization with sense cRNA probe, which largely exhibits the blue fluorescence of the counterstain, serves as control for <i>PRELI</i> mRNA detection specificity. (<b>c</b>) Northern blot results show <i>PRELI</i> expression by distinct adult mouse tissues: bone marrow (1), thymus (2), spleen (3), lymph node (4), testis (5), ovary (6), brain (7), skeletal muscle (8), heart (9), stomach (10), liver (11), lung (12) and kidney (13). Ethidium bromide-stained images of 28S and 18S ribosomal RNA bands, retained on nitrocellulose blots after capillary transfer, are shown as control for RNA sample loading (<b>d</b>) Left: Cell sorting scheme used for RNA isolation from naïve IgD⁺GL-7⁻ (1), germinal center (GC) precursors IgD⁺GL7⁺ (2), GC IgD⁻GL-7⁺ (3), and memory IgD⁻GL-7⁻ (4) B cell subsets. Right: RT-PCR results reveal <i>PRELI</i> mRNA expression by the distinct B cell subsets. Glyceraldehyde 3-phosphate dehydrogenase (<i>GAPDH</i>) mRNA amplification is presented as control of B cell subset RNA content.</p

Conditional Chr 13 <i>PRELI</i> gene Targeting.

<p>(<b>a</b>) Overall Schema <b>I</b>: WT allele depicts <i>PRELI</i> gene flanked by <i>Rab 24</i> (left) and <i>Mad3</i> (right) genes; <b>II</b>: Targeting vector construct displays relevant restrictions sites (Asc1 [A], BamH1 [B], Cla1 [C], EcoR1 [R1], Not1 [N], Pme1 [P], Sal1 [S], and Xba1 [X]). Also indicated are 5′ and 3′ arms of homology, LoxP sites, Flip recombination (Frt) sequences, and PGK-Neo and HSV-Tk selection cassettes; <b>III</b>: Floxed <i>PRELI</i> allele, after Frt-mediated removal of selection cassettes; <b>IV</b>: <i>PRELI</i> deficient allele after CD19-driven Cre recombinase (Cre) cleavage of <i>PRELI</i> exon II. Location of 5′ and 3′ external probes used in Southern Blot hybridization experiments is indicated. (<b>b</b>) PCR genotyping results of mouse litters after Chr13 <i>PRELI</i> gene targeting (Flox insertion) and Frt-mediated removal of PKG-Neo cassette but before breeding with CD19-Cre mice. Several PCR reaction products from Chr13 <i>PRELI</i>^f/f mice are compared to those of Chr13 <i>PRELI</i>^+/f and Chr13 <i>PRELI</i>^+/+ littermates. A <i>PRELI</i> gene PCR reaction from CD19Cre mouse DNA is included as an additional Chr13 <i>PRELI</i>^+/+ control. Predicted sizes of amplified DNA from PRELI^+/+ and PRELI^f/f alleles are indicated. (<b>c</b>) Southern blot genotype results of the conditionally targeted mouse strain after breeding with CD19-Cre mice. (Left) Southern blot analysis of BamH1 digested genomic DNA from WT CD19-Cre/Chr13 <i>PRELI</i>^+/+, heterozygous CD19-Cre/Chr13 <i>PRELI</i>^+/− and homozygous CD19-Cre/Chr13 <i>PRELI</i>^−/− mice hybridized to the 5′ external probe (illustrated above). Because all mouse lines tested here are in the C57BL/6 genetic background, a WT control from C57BL/6 (BL6) genomic DNA (first lane on the blot) is also included. (Right) Southern blot analysis of EcoR1 and Sal1 digested DNA from heterozygous CD19-Cre/Chr13 <i>PRELI</i>^+/−, WT CD19/Cre/Chr13 <i>PRELI</i>^+/+ and homozygous CD19-Cre/Chr13 <i>PRELI</i>^−/− mice hybridized to the 3′ external probe (illustrated above). Expected molecular sizes are also indicated.</p

Assembly of the κ PreB Receptor Requires a Vκ-like Protein Encoded by a Germline Transcript

By confining germline transcription as a byproduct of the mechanisms inherent to genetic rearrangements, the translation of respective mRNAs and their biological relevance might have been overlooked. Here we report the identification, cloning, and biochemical characterization of a human Vκ-like protein that is encoded by a germline transcript. This surrogate protein assembles with the immunoglobulin μ heavy chain at the surface of B cell progenitors and precursors to form a κ-like antigen receptor. These findings support the notion that germline transcription is not futile and stress the flexibility in eukaryotic gene usage and expression. In addition, the present study confirms the co-existence of surrogate λ and κ receptors that are proposed to work in concert to promote B lymphocyte maturation

Rational combined targeting of phosphodiesterase 4B and SYK in DLBCL

Identification of rational therapeutic targets is an important strategy to improve the cure rate of diffuse large B-cell lymphoma (DLBCL). We previously showed that inhibition of the phosphodiesterase 4B (PDE4B) unleashes cyclic-AMP (cAMP) inhibitory effects toward the PI3K/AKT pathway and induces apoptosis. These data raised important considerations as to which upstream regulators mediate cAMP inhibition of PI3K/AKT, and how identifying this signaling route could be translated into clinical initiatives. We found that in normal and malignant B cells, cAMP potently inhibit the phosphorylation and activity of the tyrosine kinase SYK. Using genetic models of gain- and loss-of-function, we demonstrated the essential role for PDE4B in controlling these effects in DLBCL. Furthermore, we used a constitutively active SYK mutant to confirm its central role in transducing cAMP effects to PI3K/AKT. Importantly, given SYK credentials as a therapeutic target in B-cell tumors, we explored the role of PDE4B in these responses. In multiple DLBCL models, we found that genetically, hence specifically, inhibiting PDE4B expression significantly improved the efficacy of SYK inhibitors. Our data defined a hitherto unknown role for cAMP in negatively regulating SYK and indicate that combined inhibition of PDE4B and SYK should be actively pursued