A time-resolved Förster resonance energy transfer assay to measure activity of the deamidase of the prokaryotic ubiquitin-like protein.

The modification of proteins in Mycobacterium tuberculosis (Mtb) by the prokaryotic ubiquitin-like protein (Pup) targets them for degradation by mycobacterial proteasomes. Although functionally similar to eukaryotic deubiquitylating enzymes, the deamidase of Pup, called Dop, has no known mammalian homologs. Because Dop is necessary for persistent infection by Mtb, its selective inhibition holds potential for tuberculosis therapy. To facilitate high-throughput screens for Dop inhibitors, we developed a time-resolved Förster resonance energy transfer (TR-FRET)-based assay for Dop function. The TR-FRET assay was successfully applied to determine the Michaelis constant for adenosine triphosphate (ATP) binding and to test the cofactor tolerance of Dop.

Chemical strategies to understand the language of ubiquitin signaling.

Ubiquitin (Ub) is a small 76 amino acid long protein that is highly conserved in all eukaryotes studied to date. In humans, more than 600 ligases are involved in the reversible modification of specific lysine side-chain amines in substrate proteins by conjugation with the C-terminal carboxylate of Ub. Initially monoubiquitylated proteins can undergo repetitive ubiquitylation starting from one of seven lysine residues or the α-amine in the first Ub to generate a variety of polyUb chains with different topologies and functions. The most well known role for protein ubiquitylation is in targeting substrates for proteolytic destruction by 26S proteasomes. However, a growing body of evidence indicates that both mono- and polyubiquitylation play proteasome-independent roles in modulating the structure, function, and localization of protein substrates. Understanding the complexity of Ub-mediated functions in our cells is a major challenge for modern biology. In addition to well-established in vivo genetic methods, biochemical and biophysical investigations of ubiquitylated proteins in vitro can shed light on the direct mechanistic roles for Ub in different contexts. Such studies have traditionally been limited by the ability to obtain sufficient quantities of homogenously ubiquitylated proteins with precisely defined linkages. This review focuses on recent advances in both synthetic and recombinant protein-based methods that have yielded access to homogenously site-specifically ubiquitylated proteins. Mechanistic studies of the roles for protein ubiquitylation and of the enzymes involved in protein deubiquitylation that are enabled by these chemical tools are highlighted.

Passively operated microfluidic device for stimulation and secretion sampling of single pancreatic islets.

A passively operated polydimethylsiloxane (PDMS) microfluidic device was designed for sampling of hormone secretions from eight individual murine pancreatic islets in parallel. Flow control was achieved using a single hand-held syringe and by exploiting inherent fluidic resistances of the microchannels (R(sampling) = 700 ± 20 kPa s mm(-3) at 37 °C). Basal (3 mM) or stimulatory (11 mM) glucose levels were applied to islets, with stimulation timing (t(stim)) minimized to 15 ± 2 s using modified reservoirs. Using enzyme-linked immunosorbent assays (ELISA) for postsampling analyses, we measured statistically equal levels of 1 h insulin secretion (1.26 ± 0.26 and 6.55 ± 1.00 pg islet(-1) min(-1), basal and stimulated; 62 islets) compared to standard, bulk sampling methods (1.01 ± 0.224 and 6.04 ± 1.53 pg islet(-1) min(-1), basal and stimulated; 200 islets). Importantly, the microfluidic platform revealed novel information on single-islet variability. Islet volume measurements with confocal reflectance microscopy revealed that insulin secretion had only limited correlation to islet volume, suggesting a more significant role for cellular architecture and paracrine signaling within the tissue. Compared to other methods using syringe pumps or electroosmotic flow control, this approach provides significant advantages in ease-of-use and device disposability, easing the burden on nonexperts.

Environmental tobacco smoke furthers progression of diabetic nephropathy.

Clinical studies suggest that smoking is a risk factor in the progression of chronic kidney disease, including diabetic nephropathy. The mechanisms involved are not completely understood. We have previously demonstrated that nicotine, one of the compounds present in large amounts in tobacco, promotes mesangial cell proliferation and fibronectin production. In this study, we hypothesized that exposure to environmental tobacco smoke (ETS) promotes the progression of diabetic nephropathy by increasing the expression of profibrotic cytokines such as transforming growth factor-beta (TGF-ß) and the extracellular matrix proteins fibronectin and collagen IV. Six-week-old diabetic (db/db) mice were divided into 2 groups. The experimental group (n = 12) was exposed to ETS at a concentration of 30 mg/m for 6 hr/d, 5 d/wk for 8 weeks. The control group (n = 8) was exposed to room air. Urine was collected before euthanasia for albumin (enzyme-linked immunosorbent assay) and creatinine measurements (mass spectrometry). After euthanasia, the kidneys were harvested for morphometric analysis and Western blot analysis. Serum was saved for cotinine measurements by enzyme-linked immunosorbent assay. ETS exposure resulted in serum levels of cotinine similar to those found in human smokers. ETS exposure for 8 weeks induced significant mesangial expansion (approximately 50% increase) that was accompanied by concomitant increases in TGF-ß and fibronectin expression (approximately 20%). However, ETS did not modify results in significant changes in urinary albumin excretion. These studies demonstrate that ETS exposure worsens the progression of diabetic nephropathy by increasing the amount of mesangial expansion and that these effects are likely mediated by increased expression of profibrotic cytokines such as TGF-ß.