Differential Proteomic Analysis of Human Erythroblasts Undergoing Apoptosis Induced by Epo-Withdrawal

The availability of Erythropoietin (Epo) is essential for the survival of erythroid progenitors. Here we study the effects of Epo removal on primary human erythroblasts grown from peripheral blood CD34+ cells. The erythroblasts died rapidly from apoptosis, even in the presence of SCF, and within 24 hours of Epo withdrawal 60% of the cells were Annexin V positive. Other classical hallmarks of apoptosis were also observed, including cytochrome c release into the cytosol, loss of mitochondrial membrane potential, Bax translocation to the mitochondria and caspase activation. We adopted a 2D DIGE approach to compare the proteomes of erythroblasts maintained for 12 hours in the presence or absence of Epo. Proteomic comparisons demonstrated significant and reproducible alterations in the abundance of proteins between the two growth conditions, with 18 and 31 proteins exhibiting altered abundance in presence or absence of Epo, respectively. We observed that Epo withdrawal induced the proteolysis of the multi-functional proteins Hsp90 alpha, Hsp90 beta, SET, 14-3-3 beta, 14-3-3 gamma, 14-3-3 epsilon, and RPSA, thereby targeting multiple signaling pathways and cellular processes simultaneously. We also observed that 14 proteins were differentially phosphorylated and confirmed the phosphorylation of the Hsp90 alpha and Hsp90 beta proteolytic fragments in apoptotic cells using Nano LC mass spectrometry. Our analysis of the global changes occurring in the proteome of primary human erythroblasts in response to Epo removal has increased the repertoire of proteins affected by Epo withdrawal and identified proteins whose aberrant regulation may contribute to ineffective erythropoiesis

HSAF-induced antifungal effects in Candida albicans through ROS-mediated apoptosis

Heat-stable antifungal factor (HSAF) belongs to polycyclic tetramate macrolactams (PTMs), which inhibits many fungal pathogens and is effective in inhibiting Candida albicans (C. albicans). In this study, we found that HSAF induced the apoptosis of C. albicans SC5314 through inducing the production of reactive oxygen species (ROS). Nevertheless, we validated the efficacy of HSAF against candidiasis caused by C. albicans in a murine model in vivo,and HSAF significantly improved survival and reduced fungal burden compared to vehicles. A molecular dynamics (MD) simulation was also investigated, revealing the theoretical binding mode of HSAF to the β-tubulin of C. albicans. This study first found PTMs-induced fungal apoptosis through ROS accumulation in C. albicans and its potential as a novel agent for fungicides

The Rho family GTPase Rif induces filopodia through mDia2

AbstractEukaryotic cells produce a variety of specialized actin-rich surface protrusions. These include filopodia—thin, highly dynamic projections that help cells to sense their external environment [1]. Filopodia consist of parallel filaments of actin, bundled by actin crosslinking proteins. The filaments are oriented with their rapidly growing “barbed” ends at the protruding tip and their slowly growing “pointed” ends at the base [2]. Extension occurs by polymerization at the tip [3] and is controlled by regulation of filament capping [4]. The Rho GTPase Cdc42 is a key mediator of filopodia formation, which it regulates through binding CRIB domain-containing effectors [2]. Cdc42 binds and activates the WASP proteins, which in turn activate the actin-nucleating complex Arp2/3 [2]. It also binds and activates IRSp53, which recruits the Ena/WASP family protein Mena [5] to the filopodial tip and protects elongating actin filaments from capping [4]. Previously, we identified another Rho family GTPase, Rif, as a potent stimulator of filopodial protrusion through a mechanism that does not require Cdc42 [6]. Here we characterize the differences between filopodia induced by these two small GTPases and show that the Rif effector in this pathway is the Diaphanous-related formin mDia2. Thus, Rif and Cdc42 represent two distinct routes to the induction of filopodia—producing structures with both shared and unique properties

Phosphorylated spots up-regulated in ESDL.

<p>Characterization of proteins fractionated by 2D-PAGE, stained with Pro-Q Diamond phosphoprotein stain and identified by Image Quant v5.2 software analysis as being hyper-phosphorylated in ESDL and then identified by Mass Spectrometry. From the 4 spots picked, 5 different proteins were identified by MS. A full list of all the peptides identified for each spot is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038356#pone.0038356.s010" target="_blank">Table S7</a>.</p

Phosphorylated spots up-regulated in SDL.

<p>Characterization of proteins fractionated by 2D-PAGE, stained with Pro-Q Diamond phosphoprotein stain and identified by Image Quant v5.2 software analysis as being hyper-phosphorylated in SDL and then identified by Mass Spectrometry. From the 9 spots picked, 9 different proteins were identified by MS. A full list of all the peptides identified for each spot is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038356#pone.0038356.s009" target="_blank">Table S6</a>.</p

Proteins with altered abundance in SDL with a change in average ratio of >2 and a <i>t</i>-test of <i>p</i><0.05.

<p>Characterization of proteins fractionated by 2D-PAGE and identified by both the DeCyder software (all gels, independent <i>t</i>-test, change in average ratio of >2 with a <i>t</i>-test of <i>p</i><0.05 ) and Mass Spectrometry, ranked in order of change in average ratio. From the 11 spots up-regulated in SDL, 11 different proteins were identified. Spot No. is number of the spot picked and shown on an SDL 2D gel (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038356#pone-0038356-g003" target="_blank">Figure 3A</a>); <i>t</i>-test and change in average ratio calculated by the DeCyder 2D Differential Analysis software when comparing the four SDL with the four ESDL 2D gels; Identified Proteins: full name of the protein identified by mass spectrometry, including isoform, as given by NCBI; NCBI Accession number and Gene ID number together with the official symbol provided by HUGO Gene Nomenclature Committee (HGNC) in brackets; Experimental (E) & Theoretical (T) Molecular mass (in kDa) and p<i>I</i>: the experimental (E) molecular mass and p<i>I</i> were determined by eye for each spot picked, whereas the theoretical (T) molecular mass and p<i>I</i> were determined using EditSeq (DNA lasergene 8) on the protein sequence of gi number identified; Number of peptide matched: total number of unique peptides matched to the protein identified; Mascot protein score: the protein score for that gi number is given. Protein score is defined as -10*Log(P), where P is the probability that the observed match is a random event. Protein scores greater than 66 (>66) are considered significant identifications (p<0.05). Where MSMS was performed, the calculated precursor ion mass and resulting peptide sequences are shown together with the corresponding ion score. The list of all the peptides identified for each spot is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038356#pone.0038356.s004" target="_blank">Table S1</a>.</p

Caspase activation after Epo removal.

<p>Western blotting (A) of total cell lysates from one independent culture, harvested after 6 hour, 12 hour and 24 hour in ESDL (+Epo) and SDL (-Epo) using an antibody against cleaved caspase 3, caspase 9, caspase 8, and hsc70 was used as a loading control. 20 µg of protein lysate was loaded in each well (B) Flow Cytometry analysis of active caspase 8, active caspase 3 and active caspase 9 for ESDL (thick black line) grown cells and SDL (dotted grey line) after 24 hours. Active caspase 9 was detected on a separate culture from the caspase 3 and caspase 9 and using a different flow cytometer.</p

Proteins with altered abundance in SDL (all gels, change in average ratio between 1.3 and 2, with a <i>t</i>-test of <i>p</i><0.05).

<p>Characterization of proteins fractionated by 2D-PAGE and identified by both the DeCyder software (all gels, independent t-test, change in average ratio between 1.3 and 2, with a <i>t</i>-test of <i>p</i><0.05 ) and Mass Spectrometry (MS), ranked in order of change in average ratio. From the 11 spots picked, 13 different proteins were identified by MS. A full list of all the peptides identified for each spot is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038356#pone.0038356.s005" target="_blank">Table S2</a>.</p

Proteins with altered abundance in SDL (3 gels, independent t-test, change in average ratio above >1.3, with a <i>t</i>-test of <i>p</i><0.05).

<p>Characterization of proteins fractionated by 2D-PAGE and identified by both the DeCyder software (3 gels, independent t-test, change in average ratio above >1.3, with a <i>t</i>-test of <i>p</i><0.05) and Mass Spectrometry (MS), ranked in order of change in average ratio. From the 7 spots picked, 7 different proteins were identified by MS. A full list of all the peptides identified for each spot is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038356#pone.0038356.s007" target="_blank">Table S4</a>.</p