Allosteric Inhibition of Ubiquitin-like Modifications by a Class of Inhibitor of SUMO-Activating Enzyme.

Ubiquitin-like (Ubl) post-translational modifications are potential targets for therapeutics. However, the only known mechanism for inhibiting a Ubl-activating enzyme is through targeting its ATP-binding site. Here we identify an allosteric inhibitory site in the small ubiquitin-like modifier (SUMO)-activating enzyme (E1). This site was unexpected because both it and analogous sites are deeply buried in all previously solved structures of E1s of ubiquitin-like modifiers (Ubl). The inhibitor not only suppresses SUMO E1 activity, but also enhances its degradation in vivo, presumably due to a conformational change induced by the compound. In addition, the lead compound increased the expression of miR-34b and reduced c-Myc levels in lymphoma and colorectal cancer cell lines and a colorectal cancer xenograft mouse model. Identification of this first-in-class inhibitor of SUMO E1 is a major advance in modulating Ubl modifications for therapeutic aims.

Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis.

We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase ɛ (Pol ɛ), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.

Proteomic profiling following immunoaffinity capture of high-density lipoprotein: association of acute-phase proteins and complement factors with proinflammatory high-density lipoprotein in rheumatoid arthritis.

OBJECTIVE: To identify protein biomarkers associated with proinflammatory high-density lipoprotein (HDL) in patients with active rheumatoid arthritis (RA) by proteomic analysis. METHODS: Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to analyze proteins associated with immunoaffinity-purified HDL from plasma obtained from 2 sets of RA patients, 1 with antiinflammatory HDL and 1 with proinflammatory HDL. Proteins were fractionated by Offgel electrophoresis and analyzed using an LC-MS/MS system equipped with a high-capacity high-performance liquid chromatography chip incorporating C18 reverse-phase trapping and analytical columns. Sandwich enzyme-linked immunosorbent assays were used to validate the association between select proteins and proinflammatory HDL in a second cohort of RA patients. RESULTS: Seventy-eight proteins were identified in the HDL complexes. The levels of 12 proteins were significantly increased in RA patients with proinflammatory HDL compared to RA patients with antiinflammatory HDL. These proteins included the acute-phase proteins apolipoprotein J, fibrinogen, haptoglobin, serum amyloid A, and complement factors (B, C3, and C9). The associations between proinflammatory HDL and 4 of the proteins were validated in a second RA cohort. CONCLUSION: Our findings indicate that proinflammatory HDL in patients with RA contains a significantly altered proteome, including increased amounts of acute-phase proteins and proteins involved in the complement cascade. These findings suggest that HDL is significantly altered in the setting of chronic inflammation in active RA, with resultant loss of its antiinflammatory function. The characterization of the biomarkers described herein may identify novel molecular connections that contribute to the higher risk of cardiovascular disease in RA patients.

Differential association of hemoglobin with proinflammatory high density lipoproteins in atherogenic/hyperlipidemic mice. A novel biomarker of atherosclerosis.

Studies in both mice and humans suggest that the anti- or proinflammatory nature of high density lipoprotein (HDL) may be a more sensitive predictor of risk for coronary heart disease events. In this study, we report the identification and characterization of two proteins (m/z 14,900 and 15,600) that are most dramatically associated with HDL in mouse models of atherosclerosis. Mass spectral analyses of proinflammatory HDL identified the two peaks to be hemoglobin (Hb) alpha and beta chains, respectively, with no apparent post-translational modification. Biochemical analysis confirmed the differential association of Hb with HDL from hyperlipidemic mice. We further show that HDL-associated Hb is predominantly in the oxyHb form with distinct physical and chemical properties. Furthermore oxyHb-containing proinflammatory HDL potently consumed nitric oxide and contracted arterial vessels ex vivo. Moreover Hb also was found differentially associated with HDL from coronary heart disease patients compared with healthy controls. Our data suggest that Hb contributes to the proinflammatory nature of HDL in mouse and human models of atherosclerosis and may serve as a novel biomarker for atherosclerosis.

Microfluidic platform for liquid chromatography-tandem mass spectrometry analyses of complex peptide mixtures.

A microfluidic chip that integrates all the fluidic components of a gradient liquid chromatography (LC) system is described. These chips were batch-fabricated on a silicon wafer using photolithographic processes and with Parylene as the main structural material. The fabricated chip includes three electrolysis-based electrochemical pumps, one for loading the sample and the other two for delivering the solvent gradient; platinum electrodes for delivering current to the pumps and establishing the electrospray potential; a low-volume static mixer; a column packed with silica-based reversed-phase support; integrated frits for bead capture; and an electrospray nozzle. The fabricated structures were able to withstand pressures in excess of 250 psi. The device was used to perform a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of a mixture of peptides from the trypsin digestion of bovine serum albumen (BSA). Gradient elution through the 1.2-cm column was performed at a flow rate of 80 nL/min. Compared to the analysis of the same sample using a commercial nanoflow LC system, the chromatographic resolution was nearly as good, and the total cycle time was significantly reduced because of the minimal volume between the pumps and the column. Results demonstrate the potential of mass-produced, low-cost microfluidic systems capable of performing LC separations for proteomics applications.

An electrochemical pumping system for on-chip gradient generation.

Within the context of microfluidic systems, it has been difficult to devise pumping systems that can deliver adequate flow rates at high pressure for applications such as HPLC. An on-chip electrochemical pumping system based on electrolysis that offers certain advantages over designs that utilize electroosmotic driven flow has been fabricated and tested. The pump was fabricated on both silicon and glass substrates using photolithography. The electrolysis electrodes were formed from either platinum or gold, and SU8, an epoxy-based photoresist, was used to form the pump chambers. A glass cover plate and a poly(dimethylsiloxane) (PDMS) gasket were used to seal the chambers. Filling of the chambers was accomplished by using a syringe to inject liquid via filling ports, which were later sealed using a glass cover plate. The current supplied to the electrodes controlled the rate of gas formation and, thus, the resulting fluid flow rate. At low backpressures, flow rates >1 microL/min have been demonstrated using <1 mW of power. Pumping at backpressures as high as 200 psi have been demonstrated, with 20 nL/min having been observed using <4 mW. By integrating two electrochemical pumps with a polymer electrospray nozzle, we have confirmed the successful generation of a solvent gradient via a mass spectrometer.