Human ESC-derived mesenchymal stem cell (MSC)-conditioned medium (CM) was previously shown to mediate cardioprotection during myocardial ischemia/reperfusion injury through large complexes of 50-100 nm. Here we show that these MSCs secreted 50- to 100-nm particles. These particles could be visualized by electron microscopy and were shown to be phospholipid vesicles consisting of cholesterol, sphingomyelin, and phosphatidylcholine. They contained coimmunoprecipitating exosome-associated proteins, e.g., CD81, CD9, and Alix. These particles were purified as a homogeneous population of particles with a hydrodynamic radius of 55-65 nm by size-exclusion fractionation on a HPLC. Together these observations indicated that these particles are exosomes. These purified exosomes reduced infarct size in a mouse model of myocardial ischemia/reperfusion injury. Therefore, MSC mediated its cardioprotective paracrine effect by secreting exosomes. This novel role of exosomes highlights a new perspective into intercellular mediation of tissue injury and repair, and engenders novel approaches to the development of biologics for tissue repair.

A conserved γ-tubulin complex–binding domain in CDK5RAP2 stimulates the microtubule-nucleating activity of γ-TuRC.

Under hypoxia, tumor cells produce a secretion that modulates their microenvironment to facilitate tumor angiogenesis and metastasis. Here, we observed that hypoxic or reoxygenated A431 carcinoma cells exhibited enhanced angiogenic and metastatic potential such as reduced cellcell and cell-extracellular matrix adhesion, increased invasiveness, and production of a secretion with increased chorioallantoic membrane angiogenic activity. Consistent with these observations, quantitative proteomics revealed that under hypoxia the tumor cells secreted proteins involved in angiogenesis, focal adhesion, extracellular matrix-receptor interaction, and immune cell recruitment. Unexpectedly, the secreted proteins were predominantly cytoplasmic and membrane proteins. Ultracentrifugation at 100,000 ؋ g precipitated 54% of the secreted proteins and enriched for many exosome-associated proteins such as the tetraspanins and Alix and also proteins with the potential to facilitate angiogenesis and metastasis. Two tetraspanins, CD9 and CD81, co-immunoprecipitated. Together, these data suggested that tumor cells secrete proteins and exosomes with the potential to modulate their microenvironment and facilitate angiogenesis and metastasis. Molecular & Cellular Proteomics 9:1085-1099, 2010.

Over the last decade a variety of MS measurements, such as H͞D exchange, collision cross sections, and electron capture dissociation (ECD), have been used to characterize protein folding in the gas phase, in the absence of solvent. To the extensive data already available on ubiquitin, here photofragmentation of its ECDreduced (M ؉ nH) (n؊1)؉• ions shows that only the 6؉ to 9؉, not the 10؉ to 13؉ ions, have tertiary noncovalent bonding; this is indicated as hydrogen bonding by the 3,050 -3,775 cm ؊1 photofragment spectrum. ECD spectra and H͞D exchange of the 13؉ ions are consistent with an all ␣-helical secondary structure, with the 11؉ and 10؉ ions sufficiently destabilized to denature small bend regions near the helix termini. In the 8؉ and 9؉ ions these terminal helical regions are folded over to be antiparallel and noncovalently bonded to part of the central helix, whereas this overlap is extended in the 7؉, 6؉, and, presumably, 5؉ ions to form a highly stable three-helix bundle. Thermal denaturing of the 7؉ to 9؉ conformers both peels and slides back the outer helices from the central one, but for the 6؉ conformer, this instead extends the protein ends away to shrink the three-helix bundle. Thus removal of H 2O from a native protein negates hydrophobic interactions, preferentially stabilizes the ␣-helical secondary structure with direct solvation of additional protons, and increases tertiary interhelix dipole-dipole and hydrogen bonding.

Recent studies suggest that the therapeutic effects of stem cell transplantation following myocardial infarction (MI) are mediated by paracrine factors. One of the main goals in the treatment of ischemic heart disease is to stimulate vascular repair mechanisms. Here, we sought to explore the therapeutic angiogenic potential of mesenchymal stem cell (MSC) secretions. Human MSC secretions were collected as conditioned medium (MSC-CM) using a clinically compliant protocol. Based on proteomic and pathway analysis of MSC-CM, an in vitro assay of HUVEC spheroids was performed identifying the angiogenic properties of MSC-CM. Subsequently, pigs were subjected to surgical left circumflex coronary artery ligation and randomized to intravenous MSC-CM treatment or non-CM (NCM) treatment for 7 days. Three weeks after MI, myocardial capillary density was higher in pigs treated with MSC-CM (645 ± 114 vs 981 ± 55 capillaries/mm(2); P = 0.021), which was accompanied by reduced myocardial infarct size and preserved systolic and diastolic performance. Intravenous MSC-CM treatment after myocardial infarction increases capillary density and preserves cardiac function, probably by increasing myocardial perfusion.

No abstract

Asparaginyl endopeptidases (AEPs) are cysteine proteases which break Asx (Asn/Asp)-Xaa bonds in acidic conditions. Despite sharing a conserved overall structure with AEPs, certain plant enzymes such as butelase 1 act as a peptide asparaginyl ligase (PAL) and catalyze Asx-Xaa bond formation in near-neutral conditions. PALs also serve as macrocyclases in the biosynthesis of cyclic peptides. Here, we address the question of how a PAL can function as a ligase rather than a protease. Based on sequence homology of butelase 1, we identified AEPs and PALs from the cyclic peptideproducing plants Viola yedoensis (Vy) and Viola canadensis (Vc) of the Violaceae family. Using a crystal structure of a PAL obtained at 2.4-Å resolution coupled to mutagenesis studies, we discovered ligase-activity determinants flanking the S1 site, namely LAD1 and LAD2 located around the S2 and S1′ sites, respectively, which modulate ligase activity by controlling the accessibility of water or amine nucleophile to the S-ester intermediate. Recombinantly expressed VyPAL1-3, predicted to be PALs, were confirmed to be ligases by functional studies. In addition, mutagenesis studies on VyPAL1-3, VyAEP1, and VcAEP supported our prediction that LAD1 and LAD2 are important for ligase activity. In particular, mutagenesis targeting LAD2 selectively enhanced the ligase activity of VyPAL3 and converted the protease VcAEP into a ligase. The definition of structural determinants required for ligation activity of the asparaginyl ligases presented here will facilitate genomic identification of PALs and engineering of AEPs into PALs. peptide ligase | data mining | ligase-activity determinant

A mass difference between the measured molecular weight of a protein and that of its DNA-predicted sequence indicates sequence errors and͞or posttranslational modifications. In the top-down mass spectrometry approach, the measured molecular ion is dissociated, and these fragment masses are matched against those predicted from the protein sequence to restrict the locations of the errors͞modifications. The proportion of the ion's interresidue bonds that are cleaved determines the specificity of such locations; previously, ubiquitin (76 residues) was the largest for which all such bonds were dissociated. Now, cleavages are achieved for carbonic anhydrase at 250 of the 258 interresidue locations. Cleavages of three spectra would define posttranslational modifications at 235 residues to within one residue. For 24 of the 34 possible phosphorylation sites, the cleavages of one spectrum would delineate exactly all ؊PO 3H substitutions. This result has been achieved with electron-capture dissociation by minimizing the further cleavage of primary product ions and by denaturing the tertiary noncovalent bonding of the molecular ions under a variety of conditions. T he explosive growth in the applications of mass spectrometry (MS) to proteomics can be traced back to the discovery four decades ago by Biemann, who showed that the mass spectrum of a peptide can directly indicate its sequence of amino acids (1). This approach was extended to larger peptides (2), to mixtures using MS͞MS (3), and to proteins (4-10), even at the 10 Ϫ17 mol level (11,12). For ''top down'' protein MS͞MS (7-10), masses of 7601.5 and 7865.7 Da for two fragment ions from carbonic anhydrase indicate the presence of Tyr and Thr (Tyr, 163.06 Da; Thr, 101.05 Da), matching the DNA-predicted at this mass distance from the C terminus (7). For the more difficult structural proteomics problem of characterizing posttranslational modifications, a molecular ion mass 80 Da higher than predicted could indicate phosphorylation (HPO 3 , 79.97 Da; ref. 13); if the fragment mass ϳ7601.5 is present, but 7865.7 is replaced by ϳ7946, the ϩ80-Da modification is at . An intermediate cleavage is necessary to differentiate these; a 7764.6-Da peak would show that Thr-191 is modified. This limitation in the proportion of interresidue bonds cleaved has been far more severe for such larger proteins. Top-down Fourier transfrom MS of the 8.6-kDa ubiquitin gave masses representing all interresidue cleavages, making possible its complete sequencing de novo (14). However, MS͞MS of ␤-casein (3 variants, Ϸ24 kDa) identified the phosphorylation site at Ser-15, limited three more sites to five locations, but only restricted the last site to four possibilities (the protein contains 27 possible sites; ref. 13). Many MS͞MS spectra of carbonic anhydrase (259 residues) combined gave cleavages between 135 different residue pairs, by far the most reported for any protein ion (15). Improvements here to electron capture dissociation (ECD; result in the cleavage of 250 of the 258 interresidue bonds...