Proteome Changes during Transition from Human Embryonic to Vascular Progenitor Cells

Human embryonic stem cells (hESCs) are promising in regenerative medicine (RM) due to their differentiation plasticity and proliferation potential. However, a major challenge in RM is the generation of a vascular system to support nutrient flow to newly synthesized tissues. Here we refined an existing method to generate tight vessels by differentiating hESCs in CD34⁺ vascular progenitor cells using chemically defined media and growth conditions. We selectively purified these cells from CD34^– outgrowth populations also formed. To analyze these differentiation processes, we compared the proteomes of the hESCs with those of the CD34⁺ and CD34^– populations using high resolution mass spectrometry, label-free quantification, and multivariate analysis. Eighteen protein markers validate the differentiated phenotypes in immunological assays; nine of these were also detected by proteomics and show statistically significant differential abundance. Another 225 proteins show differential abundance between the three cell types. Sixty-three of these have known functions in CD34⁺ and CD34^– cells. CD34⁺ cells synthesize proteins implicated in endothelial cell differentiation and smooth muscle formation, which support the bipotent phenotype of these progenitor cells. CD34^– cells are more heterogeneous synthesizing muscular/osteogenic/chondrogenic/adipogenic lineage markers. The remaining >150 differentially abundant proteins in CD34⁺ or CD34^– cells raise testable hypotheses for future studies to probe vascular morphogenesis

Proteome Changes during Transition from Human Embryonic to Vascular Progenitor Cells

Human embryonic stem cells (hESCs) are promising in regenerative medicine (RM) due to their differentiation plasticity and proliferation potential. However, a major challenge in RM is the generation of a vascular system to support nutrient flow to newly synthesized tissues. Here we refined an existing method to generate tight vessels by differentiating hESCs in CD34⁺ vascular progenitor cells using chemically defined media and growth conditions. We selectively purified these cells from CD34^– outgrowth populations also formed. To analyze these differentiation processes, we compared the proteomes of the hESCs with those of the CD34⁺ and CD34^– populations using high resolution mass spectrometry, label-free quantification, and multivariate analysis. Eighteen protein markers validate the differentiated phenotypes in immunological assays; nine of these were also detected by proteomics and show statistically significant differential abundance. Another 225 proteins show differential abundance between the three cell types. Sixty-three of these have known functions in CD34⁺ and CD34^– cells. CD34⁺ cells synthesize proteins implicated in endothelial cell differentiation and smooth muscle formation, which support the bipotent phenotype of these progenitor cells. CD34^– cells are more heterogeneous synthesizing muscular/osteogenic/chondrogenic/adipogenic lineage markers. The remaining >150 differentially abundant proteins in CD34⁺ or CD34^– cells raise testable hypotheses for future studies to probe vascular morphogenesis

MOESM1 of Large-scale production of a thermostable Rhodothermus marinus cellulase by heterologous secretion from Streptomyces lividans

Additional file 1: Figure S1. Amino acid sequence of cellulase A (CelA). Figure S2. pIJ486_Vsi-celA plasmid. Table S1. Annotated secreted cellulase-related hydrolases in the S. lividans TK24 proteome

MOESM4 of Comprehensive subcellular topologies of polypeptides in Streptomyces

Additional file 4: Table S4. Conflicts in protein topology between Uniprot and SToPSdb

MOESM1 of Comprehensive subcellular topologies of polypeptides in Streptomyces

Additional file 1: Table S1. Re-annotated protein names and Identifiers for the proteins included in SToPSdb

MOESM5 of Comprehensive subcellular topologies of polypeptides in Streptomyces

Additional file 5: Table S5. Comparison of SToPSdb and LocateP topological annotation

MOESM2 of Comprehensive subcellular topologies of polypeptides in Streptomyces

Additional file 2: Table S2. Additional examples of protein description re-annotation in SToPSdb

MOESM6 of Comprehensive subcellular topologies of polypeptides in Streptomyces

Additional file 6: Table S6. Secretion system components in S. lividans TK24

MOESM3 of Comprehensive subcellular topologies of polypeptides in Streptomyces

Additional file 3: Table S3. Protein topology nomenclature

Data_Sheet_1_Multi-Omics and Targeted Approaches to Determine the Role of Cellular Proteases in Streptomyces Protein Secretion.PDF

<p>Gram-positive Streptomyces bacteria are profuse secretors of polypeptides using complex, yet unknown mechanisms. Many of their secretory proteins are proteases that play important roles in the acquisition of amino acids from the environment. Other proteases regulate cellular proteostasis. To begin dissecting the possible role of proteases in Streptomyces secretion, we applied a multi-omics approach. We probed the role of the 190 proteases of Streptomyces lividans strain TK24 in protein secretion in defined media at different stages of growth. Transcriptomics analysis revealed transcripts for 93% of these proteases and identified that 41 of them showed high abundance. Proteomics analysis identified 57 membrane-embedded or secreted proteases with variations in their abundance. We focused on 17 of these proteases and putative inhibitors and generated strains deleted of their genes. These were characterized in terms of their fitness, transcriptome and secretome changes. In addition, we performed a targeted analysis in deletion strains that also carried a secretion competent mRFP. One strain, carrying a deletion of the gene for the regulatory protease FtsH, showed significant global changes in overall transcription and enhanced secretome and secreted mRFP levels. These data provide a first multi-omics effort to characterize the complex regulatory mechanisms of protein secretion in Streptomyces lividans and lay the foundations for future rational manipulation of this process.</p