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Protein glycosylation, one of the most heterogeneous post-translational modifications, can play a major role in cellular signal transduction and disease progression. Traditional mass spectrometry (MS)-based large-scale glycoprotein sequencing studies heavily rely on identifying enzymatically released glycans and their original peptide backbone separately, as there is no efficient fragmentation method to produce unbiased glycan and peptide product ions simultaneously in a single spectrum and can be conveniently applied to high throughput glycoproteome characterization, especially for N-glycopeptides which can have much more branched glycan side chains than relatively less complex O-linked glycans. In this study a re-defined electron-transfer/higher-energy collision dissociation (EThcD) fragmentation scheme is applied to incorporate both glycan and peptide fragments in one single spectrum, enabling complete information to be gathered and great microheterogeneity details to be revealed. Fetuin was first utilized to prove the applicability with 19 glycopeptides and corresponding 5 glycosylation sites identified. Subsequent experiments tested its utility for human plasma N-glycoproteins. Large-scale studies explored N-glycoproteomics in rat carotid over the course of restenosis progression to investigate potential role of glycosylation. The integrated fragmentation scheme provides a powerful tool for the analysis of intact N-glycopeptides and N-glycoproteomics. We also anticipate this approach can be readily applied to large-scale O-glycoproteome characterization.

The retinal phosphodiesterase (PDE6) inhibitory ␥-subunit (PDE␥) plays a central role in vertebrate phototransduction through alternate interactions with the catalytic ␣␤-subunits of PDE6 and the ␣-subunit of transducin (␣t). Detailed structural analysis of PDE␥ has been hampered by its intrinsic disorder. We present here the NMR solution structure of PDE␥, which reveals a loose fold with transient structural features resembling those seen previously in the x-ray structure of PDE␥46-87 when bound to ␣t in the transitionstate complex. NMR mapping of the interaction between PDE␥46-87 and the chimeric PDE5/6 catalytic domain confirmed that Cterminal residues 74 -87 of PDE␥ are involved in the association and demonstrated that its W70 indole group, which is critical for subsequent binding to ␣t, is left free at this stage. These results indicate that the interaction between PDE␥ and ␣t during the phototransduction cascade involves the selection of preconfigured transient conformations.NMR spectroscopy ͉ protein recognition ͉ transient structure ͉ visual transduction P hototransduction, the primary event of vision is critically regulated by the ␥-subunit of cGMP phosphodiesterase (PDE␥). In the dark, by binding tightly to the catalytic ␣␤-subunits of cGMP PDE (PDE6) PDE␥ keeps PDE6 inactive; yet upon photoexcitation, interaction of PDE␥ with the ␣-subunit of activated transducin (␣ t ) relieves its inhibitory constraint on PDE6. For termination of phototransduction, PDE␥ interacts with both ␣ t and RGS9, a regulator of G protein signaling, to accelerate ␣ t GTPase activity (1). Beyond phototransduction, the importance of PDE␥ in photoreceptor cell viability is demonstrated by rapid retinal degeneration in the PDE␥-knockout mouse (2), and growing evidence suggests that PDE␥ also interacts with some signaling proteins in nonphototransduction pathways (3).Biochemical studies have shown that the central polycationic region (residues 24-45) and the C-terminal region of PDE␥ constitute two distinct sites of interactions with ␣ t or PDE6 (4). The crystal structure of a ternary complex representing a partial model for the GTPase-activating protein (GAP) complex, consisting of a fragment of PDE␥ (residues 46-87), ␣ t chimera (␣ t/i1 ), and the catalytic core of RGS9, revealed that the C-terminal region of PDE␥ contains three discontinuous helices (5). PDE␥ 46-87 interacts with ␣ t in a GTP-dependent manner, mainly through residues in these helices or their linkers, and the indole side chain of W70 plays the key role in this interaction. Residue V66 of PDE␥ interacts with RGS9, which further tightens the association between ␣ t and RGS9. The C-terminal region of PDE␥ also interacts with the catalytic domain of PDE6; however, studies have suggested that this interaction involves the 11 C-terminal residues of PDE␥, a region distinct from the ␣ t interaction site (6).It has been demonstrated that PDE␥ belongs to the growing family of intrinsically disordered proteins (IDPs) (7,8). Presumably, the inherent structural plasticity of...

BackgroundIntimal hyperplasia is a common cause of many vasculopathies. There has been a recent surge of interest in the bromo and extra-terminal (BET) epigenetic “readers” including BRD4 since the serendipitous discovery of JQ1(+), an inhibitor specific to the seemingly undruggable BET bromodomains. The role of the BET family in the development of intimal hyperplasia is not known.MethodsWe investigated the effect of BET inhibition on intimal hyperplasia using a rat balloon angioplasty model.ResultsWhile BRD4 was dramatically up-regulated in the rat and human hyperplastic neointima, blocking BET bromodomains with JQ1(+) diminished neointima in rats. Knocking down BRD4 with siRNA, or treatment with JQ1(+) but not the inactive enantiomer JQ1(−), abrogated platelet-derived growth factor (PDGF-BB)-stimulated proliferation and migration of primary rat aortic smooth muscle cells. This inhibitory effect of JQ1(+) was reproducible in primary human aortic smooth muscle cells. In human aortic endothelial cells, JQ1(+) prevented cytokine-induced apoptosis and impairment of cell migration. Furthermore, either BRD4 siRNA or JQ1(+) but not JQ1(−), substantially down-regulated PDGF receptor-α which, in JQ1(+)-treated arteries versus vehicle control, was also reduced.ConclusionsBlocking BET bromodomains mitigates neointima formation, suggesting an epigenetic approach for effective prevention of intimal hyperplasia and associated vascular diseases.