Proteomic Landscape and Deduced Functions of the Cardiac 14-3-3 Protein Interactome

Rationale: The 14-3-3 protein family is known to interact with many proteins in non-cardiac cell types to regulate multiple signaling pathways, particularly those relating to energy and protein homeostasis; and the 14-3-3 network is a therapeutic target of critical metabolic and proteostatic signaling in cancer and neurological diseases. Although the heart is critically sensitive to nutrient and energy alterations, and multiple signaling pathways coordinate to maintain the cardiac cell homeostasis, neither the structure of cardiac 14-3-3 protein interactome, nor potential functional roles of 14-3-3 protein–protein interactions (PPIs) in heart has been explored. Objective: To establish the comprehensive landscape and characterize the functional role of cardiac 14-3-3 PPIs. Methods and Results: We evaluated both RNA expression and protein abundance of 14-3-3 isoforms in mouse heart, followed by co-immunoprecipitation of 14-3-3 proteins and mass spectrometry in left ventricle. We identified 52 proteins comprising the cardiac 14-3-3 interactome. Multiple bioinformatic analyses indicated that more than half of the proteins bound to 14-3-3 are related to mitochondria; and the deduced functions of the mitochondrial 14-3-3 network are to regulate cardiac ATP production via interactions with mitochondrial inner membrane proteins, especially those in mitochondrial complex I. Binding to ribosomal proteins, 14-3-3 proteins likely coordinate protein synthesis and protein quality control. Localizations of 14-3-3 proteins to mitochondria and ribosome were validated via immunofluorescence assays. The deduced function of cardiac 14-3-3 PPIs is to regulate cardiac metabolic homeostasis and proteostasis. Conclusions: Thus, the cardiac 14-3-3 interactome may be a potential therapeutic target in cardiovascular metabolic and proteostatic disease states, as it already is in cancer therapy

Temperatura do ar: dados climáticos relativos ao mês de março de 1989

Os dados foram recolhidos e registados pelo técnico da ESACB João Nunes sob a supervisão da Prof.ª Maria do Carmo Horta Monteiro.Dados climáticos relativos ao ano de 1989, recolhidos e registados no Posto Meteorológico da Escola Superior Agrária do Instituto Politécnico de Castelo Branco

Examination of known somatic cell B-Myb target gene expression patterns following knockdown of B-Myb in ESCs.

<p>A) Western blot and graphic presentation of Cyclin B1 (Ccnb1) protein expression following knockdown of B-MYB by shRNAs 1, 2 and 5. B) RNA (left) and protein (right) analysis of the B-MYB target gene Polo-like kinase 1 (Plk1) following Bγ-Myb KD with shRNA1 and shRNA5. The effects of B-MYB KD on Plk1 RNA were transient; however, the loss of RNA led to a significant and sustained decrease in its protein abundance. C) Western blot and bar graph showing the abundance of selected proteins following knockdown of B-MYB and CCNB1 using shRNAs and microRNAs, respectively. D) Reduced expression of CCNB1 could not mimic the cell cycle related defects associated with B-MYB deficiency, such as monopolar or multipolar centrosomes as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042350#pone-0042350-g001" target="_blank">Figure 1</a>. Ctl – control; KD – Knockdown; NS- non-silencing. *, p<0.05. Data are expressed as mean ± standard deviation (SD).</p

Biological processes and pathways associated with B-MYB deficiency.

<p>A total 18,097 genes were ranked by fold-change of gene expression between control and B-MYB knockdown in mouse ESCs based on microarray experiments (see Methods). Ranked genes were analyzed using GSEA based on GO biological processes. The biological processes in the table represents enriched ones with P value of 0 for under-expressed genes by B-MYB knockdown.</p

B-MYB function and phosphorylation in pluripotent stem cells.

<p>A) Western blot showing presence of B-MYB in ESCs (R1) and iPSCs (2D4). B) Graphic representation of the average number of cells present within individual ESC colonies (n = 40 colonies/group) 48 hours after nucleofection with control or targeting shRNA1 vectors to B-MYB. C) Typical image of pulsed BrdU incorporation in control and B-MYB deficient ESCs, showing decreased BrdU incorporation in B-Myb knockdown (KD) cells. D) DNA content measured by flow cytometry of non-synchronized fixed ES cells stained with propidium iodide after nucleofection with shRNA constructs. At 48 hours post-nucleofection, including 24 hours of selection with puromycin, a shift in the DNA content to a G2/M prevalent and aneuploid (8N) state (see arrows in inset) can be detected. E) Quantification of the cell cycle distributions following knockdown (KD) of B-MYB. The number of cells in S phase decreased concomitant to an increase in the number of cells in G2/M relative to controls and a significant increase in octoploidy (8N). F) Representative mitotic cells stained with DAPI (blue), α-tubulin (green) and γ-tubulin (red) are shown. In these experiments, mitotic spindle and centrosome defects are readily observed in cells lacking B-Myb. A quantitative assessment of spindle defects detected in B-Myb deficient cells (n = 3, 100 cells/group) is shown in the graph below. G) Images of immunostained ESC colonies with antibodies specific for phosphorylated forms of B-MYB. It is noteworthy that all mitotic cells show phosphorylation at Thr490 or Thr497. H) Flow cytometry analysis of mouse ESCs showing that phosphorylated forms of B-MYB are present only in the G2/M phases of the cell cycle (see boxed region). The number of cells present in the boxed regions correlates directly with the number of red mitotic cells shown in the inset of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042350#pone-0042350-g001" target="_blank">Figure 1G</a>. Size marker = 15 µm. *, p<0.05. Data are expressed as mean ± standard deviation (SD).</p

Global network analysis showing the distribution of connections per node in the co-expression networks.

<p>A) Interactome showing hub genes and connectivities among hubs of selected pluripotency, cell cycle and epigenetic regulator-associated genes in control (Ctl) and B-Myb knockdown (KD) cells. The size of each circle is directly proportional to the number of links (connectivity) for the indicated hub gene, and lines between genes are indicative of the number of overlapping links. As shown in this figure, B-Myb, Eed and Sall4 are major hub genes in control cells that have overlapping links with over 160 genes. Following knockdown of B-MYB, all of major hub genes either become minor hubs or lose all connectivity (shown in red). B) Based on the analysis, Sall4, a transcription factor found to be central to the control ESC network, loses all connectivity in B-MYB deficient ESCs (4A); but in response to the loss of B-MYB, Sall4 transcript and protein abundance increase significantly, suggesting that the up-regulation of this gene is a compensatory response to the loss of B-MYB.</p