Activation of glycolytic genes by HIF-1 is considered critical for metabolic adaptation to hypoxia through increased conversion of glucose to pyruvate and subsequently to lactate. We found that HIF-1 also actively suppresses metabolism through the tricarboxylic acid cycle (TCA) by directly trans-activating the gene encoding pyruvate dehydrogenase kinase 1 (PDK1). PDK1 inactivates the TCA cycle enzyme, pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA. Forced PDK1 expression in hypoxic HIF-1alpha null cells increases ATP levels, attenuates hypoxic ROS generation, and rescues these cells from hypoxia-induced apoptosis. These studies reveal a hypoxia-induced metabolic switch that shunts glucose metabolites from the mitochondria to glycolysis to maintain ATP production and to prevent toxic ROS production.

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Induction of a pluripotent state in somatic cells through nuclear reprogramming has ushered in a new era of regenerative medicine. Heterogeneity and varied differentiation potentials among induced pluripotent stem cell (iPSC) lines are, however, complicating factors that limit their usefulness for disease modeling, drug discovery, and patient therapies. Thus, there is an urgent need to develop nonmutagenic rapid throughput methods capable of distinguishing among putative iPSC lines of variable quality. To address this issue, we have applied a highly specific chemoproteomic targeting strategy for de novo discovery of cell surface N-glycoproteins to increase the knowledge-base of surface exposed proteins and accessible epitopes of pluripotent stem cells. We report the identification of 500 cell surface proteins on four embryonic stem cell and iPSCs lines and demonstrate the biological significance of this resource on mouse fibroblasts containing an oct4-GFP expression cassette that is active in reprogrammed cells. These results together with immunophenotyping, cell sorting, and functional analyses demonstrate that these newly identified surface marker panels are useful for isolating iPSCs from heterogeneous reprogrammed cultures and for isolating functionally distinct stem cell subpopulations. Molecular & Cellular

Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes encoding proteins that enable cells to adapt to reduced O 2 availability. Proteins encoded by HIF-1 target genes play a central role in mediating physiological processes that are dysregulated in cancer and heart disease. These diseases are also characterized by increased production of cyclic adenosine monophosphate (cAMP), the allosteric activator of cAMP-dependent protein kinase A (PKA). Using GSTpulldown, coimmunoprecipitation and mass spectrometry analyses, we demonstrated that PKA interacts with HIF-1α in HeLa cervical carcinoma cells and rat cardiomyocytes. PKA phosphorylated Thr 63 and Ser 692 on HIF-1α in vitro and enhanced HIF transcriptional activity and target gene expression in HeLa cells and rat cardiomyocytes. PKA inhibited the proteasomal * Corresponding author. gsemenza@jhmi.edu. Author contributions: JWB and GLS designed the study, analyzed data, and wrote the paper. IT, RJH, VV and JVE performed MS/MS analyses of HIF-1α-interacting proteins. LD and RNC performed MS/MS analyses of phosphorylated HIF-1α. FW and DLK provided primary neonatal rat cardiomyocytes. All authors reviewed the results and approved the final version of the manuscript. Competing interests:The authors declare that they have no competing interests. Data and materials availability:The HIF-1a interacting protein and phosphorylated HIF-1α residues mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE (40) partner repository with the dataset identifiers PXD003792 and PXD003795, respectively. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript degradation of HIF-1α in an O 2 -independent manner that required the phosphorylation of Thr 63 and Ser 692 and was not affected by prolyl hydroxylation. PKA also stimulated the binding of the coactivator p300 to HIF-1α to enhance its transcriptional activity and counteracted the inhibitory effect of asparaginyl hydroxylation on the association of p300 with HIF-1α. Furthermore, increased cAMP concentrations enhanced the expression of HIF target genes encoding CD39 and CD73, which are enzymes that convert extracellular ATP to adenosine, a molecule that enhances tumor immunosuppression and reduces heart rate and contractility. These data link stimuli that promote cAMP signaling, HIF-1α-dependent changes in gene expression, and increased adenosine, all of which contribute to the pathophysiology of cancer and heart disease.

High abundance proteins in serum and plasma (e.g., albumin) are routinely removed during proteomic sample processing as they can mask lower abundance proteins and peptides of biological/clinical interest. A common method of albumin depletion is based on immunoaffinity capture, and many immunoaffinity devices are designed for multiple uses. In this case, it is critical that the albumin captured on the affinity matrix is stripped from the column prior to regeneration of the matrix and processing of subsequent samples, to ensure no carryover and that maximal binding sites are available for subsequent samples. The current study examines the ability of a manufacturer’s protocol to remove the proteins and peptides captured by an immunoaffinity spin column. The data presented in the current work illustrate the difficulty in completely removing albumin from the immunoaffinity device, and consequently, may explain the variability and decreased efficiency shown for this device in previous studies. In summary, the current data present important considerations for the implementation of multiple-use immunoaffinity devices for processing subsequent clinical samples in a proteomic workflow.

Cellular contractility is governed by a control system of proteins that integrates internal and external cues to drive diverse shape change processes. This contractility controller includes myosin II motors, actin crosslinkers and protein scaffolds, which exhibit robust and cooperative mechanoaccumulation. However, the biochemical interactions and feedback mechanisms that drive the controller remain unknown. Here, we use a proteomics approach to identify direct interactors of two key nodes of the contractility controller in the social amoeba Dictyostelium discoideum: the actin crosslinker cortexillin I and the scaffolding protein IQGAP2. We highlight several unexpected proteins that suggest feedback from metabolic and RNA-binding proteins on the contractility controller. Quantitative in vivo biochemical measurements reveal direct interactions between myosin II and cortexillin I, which form the core mechanosensor. Furthermore, IQGAP1 negatively regulates mechanoresponsiveness by competing with IQGAP2 for binding the myosin II-cortexillin I complex. These myosin II-cortexillin I-IQGAP2 complexes are pre-assembled into higher-order mechanoresponsive contractility kits (MCKs) that are poised to integrate into the cortex upon diffusional encounter coincident with mechanical inputs. This article has an associated First Person interview with the first author of the paper.

Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function, and one of the best examples of endogenous repair mechanisms involves skeletal muscle. Although the molecular mechanisms that regulate the differentiation of satellite cells and myoblasts toward myofibers are not fully understood, cell surface proteins that sense and respond to their environment play an important role. The cell surface capturing technology was used here to uncover the cell surface N-linked glycoprotein subproteome of myoblasts and to identify potential markers of myoblast differentiation. 128 bona fide cell surface-exposed N-linked glycoproteins, including 117 transmembrane, four glycosylphosphatidylinositol-anchored, five extracellular matrix, and two membrane-associated proteins were identified from mouse C2C12 myoblasts. The data set revealed 36 cluster of differentiation-annotated proteins and confirmed the occupancy for 235 N-linked glycosylation sites. The identification of the N-glycosylation sites on the extracellular domain of the proteins allowed for the determination of the orientation of the identified proteins within the plasma membrane. One glycoprotein transmembrane orientation was found to be inconsistent with Swiss-Prot annotations, whereas ambiguous annotations for 14 other proteins were resolved. Several of the identified N-linked glycoproteins, including aquaporin-1 and β-sarcoglycan, were found in validation experiments to change in overall abundance as the myoblasts differentiate toward myotubes. Therefore, the strategy and data presented shed new light on the complexity of the myoblast cell surface subproteome and reveal new targets for the clinically important characterization of cell intermediates during myoblast differentiation into myotubes.

The current study used three different proteomic strategies, which differed by their extent of intact protein separation, to examine the proteome of a pluripotent mouse embryonic stem cell line, R1.Proteins from whole-cell lysates were subjected either to 2-D-LC, or 1-DE, or were unfractionated prior to enzymatic digestion and subsequent analysis by MS. The results yielded 1895 identified nonredundant proteins and, for 128 of these, the specific isoform could be determined based on detection of an isoform-specific peptide. When compared with two previously published proteomic studies that used the same cell line, the current study reveals 612 new proteins. The realization of stem cell therapy depends, in part, on understanding and manipulating mechanisms necessary for the maintenance of pluripotency as well as differentiation into specific cell types. Knowing the genes and proteins that play essential roles in these processes is an important part of understanding stem cell biology and developing viable therapies. Therefore, studies that characterize the proteome of pluripotent cells will benefit the stem cell community. Toward that end, the current study used three different proteomic strategies, which differed by their extent of intact protein separation, to examine the proteome of a pluripotent mouse embryonic stem (ES) cell line, R1. This work complements previous proteomic studies of the R1 proteome by our lab [1], which used 2-DE, and Graumann et al. [2], which used subcellular fractionation followed by 1-DE for protein separation and isoelectric focusing for peptide separation. NIH Public Access Author ManuscriptProteomics. Author manuscript; available in PMC 2010 September 7. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript transcripts to Oct4, sex-determining region Y-box 2, Nanog, Zfp42, and either weak or no expression of transcript markers of differentiation (Brachyury, CoupTF) (data not shown).Protein from whole-cell lysates were subjected either to 2-D-LC; (separation by pI and hydrophobicity), 1-DE (separation by molecular mass), or were unfractionated (UF; i.e. shotgun approach) prior to enzymatic digestion and subsequent analysis by MS (Fig. 1). Detailed methods are provided in the Supporting Information. Peptides from the 1-DE (n = 20 bands) and UF samples (n = 2 replicates) were analyzed on an Agilent 1200 nanoLC system (Agilent, Santa Clara, CA, USA) connected to an LTQ-Orbitrap mass spectrometer (Thermo). 2-D-LC samples (n = 185 fractions) were analyzed on an Agilent 1100 nano-LC system connected to an LTQ mass spectrometer (Thermo The ProteinProphet interact-prot.xml result files were input into ProteinCenter (Proxeon Bioinformatics, Odense, Denmark) and filtered to display proteins with protein probability scores p>0.9 (corresponding to false discovery rate ~1.0%), which were identified by two or more unique peptides. To remove redundancy in protein identifications, proteins were grouped according to "indistinguishable proteins," which resulted in 1895 pro...