Agonist-biased trafficking of somatostatin receptor 2A in enteric neurons.

Somatostatin (SST) 14 and SST 28 activate somatostatin 2A receptors (SSTR2A) on enteric neurons to control gut functions. SST analogs are treatments of neuroendocrine and bleeding disorders, cancer, and diarrhea, with gastrointestinal side effects of constipation, abdominal pain, and nausea. How endogenous agonists and drugs differentially regulate neuronal SSTR2A is unexplored. We evaluated SSTR2A trafficking in murine myenteric neurons and neuroendocrine AtT-20 cells by microscopy and determined whether agonist degradation by endosomal endothelin-converting enzyme 1 (ECE-1) controls SSTR2A trafficking and association with ß-arrestins, key regulators of receptors. SST-14, SST-28, and peptide analogs (octreotide, lanreotide, and vapreotide) stimulated clathrin- and dynamin-mediated internalization of SSTR2A, which colocalized with ECE-1 in endosomes and the Golgi. After incubation with SST-14, SSTR2A recycled to the plasma membrane, which required active ECE-1 and an intact Golgi. SSTR2A activated by SST-28, octreotide, lanreotide, or vapreotide was retained within the Golgi and did not recycle. Although ECE-1 rapidly degraded SST-14, SST-28 was resistant to degradation, and ECE-1 did not degrade SST analogs. SST-14 and SST-28 induced transient interactions between SSTR2A and ß-arrestins that were stabilized by an ECE-1 inhibitor. Octreotide induced sustained SSTR2A/ß-arrestin interactions that were not regulated by ECE-1. Thus, when activated by SST-14, SSTR2A internalizes and recycles via the Golgi, which requires ECE-1 degradation of SST-14 and receptor dissociation from ß-arrestins. After activation by ECE-1-resistant SST-28 and analogs, SSTR2A remains in endosomes because of sustained ß-arrestin interactions. Therapeutic SST analogs are ECE-1-resistant and retain SSTR2A in endosomes, which may explain their long-lasting actions.

Profiling protease activities by dynamic proteomics workflows.

Proteases play prominent roles in many physiological processes and the pathogenesis of various diseases, which makes them interesting drug targets. To fully understand the functional role of proteases in these processes, it is necessary to characterize the target specificity of the enzymes, identify endogenous substrates and cleavage products as well as protease activators and inhibitors. The complexity of these proteolytic networks presents a considerable analytic challenge. To comprehensively characterize these systems, quantitative methods that capture the spatial and temporal distributions of the network members are needed. Recently, activity-based workflows have come to the forefront to tackle the dynamic aspects of proteolytic processing networks in vitro, ex vivo and in vivo. In this review, we will discuss how mass spectrometry-based approaches can be used to gain new insights into protease biology by determining substrate specificities, profiling the activity-states of proteases, monitoring proteolysis in vivo, measuring reaction kinetics and defining in vitro and in vivo proteolytic events. In addition, examples of future aspects of protease research that go beyond mass spectrometry-based applications are given.

Protease- and acid-catalyzed labeling workflows employing (18)O-enriched water.

Stable isotopes are essential tools in biological mass spectrometry. Historically, (18)O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes(1-3). With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of (18)O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao(4), Miyagi and Rao(5) and Ye et al.(6)). (18)O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques(7). Depending on the protease utilized, (18)O-labeling can result in the incorporation of up to two (18)O-atoms in the C-terminal carboxyl group of the cleavage product(3). The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction(8). In our PALeO (protease-assisted labeling employing (18)O-enriched water) adaptation of enzymatic (18)O-labeling, we utilized 50% (18)O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases(9) and monitor proteolytic reactions(10-11). Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second (18)O-atom. Such "double-labeling" enzymes can be used for postdigestion (18)O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing (18)O-enriched water beyond enzymes and uses acidic pH conditions to introduce (18)O-stable isotope signatures into peptides.

High-resolution gas chromatography retention data as a basis for estimation of the octanol-water distribution coefficients (Kow) of PCB: the effect of experimental conditions.

The semi-experimental approach to approximating physicochemical data relevant to environmental distribution (vapor pressure and gas-octanol distribution) by correlation with gas chromatography (GC) retention data has been extended to the determination of Kow values. We estimated Kow values >10(4) for polychlorinated biphenyls (PCB), which are often derived by liquid chromatography, by correlation with gas chromatographic retention data. Selecting a set of reference compounds with known Kow values for relative retention time (RRT) correlation enables easy and accurate semi-empirical calculation of further Kow values for a given group of congeners. The RRT/log Kow correlation is validated in this paper with regard to the following gas chromatographic conditions: (1) isothermal versus temperature-programmed elution, (2) the possible effect of the polarity of the stationary phase, and (3) the effect of the format of the standardized GC retention data. The advantages of our Kow(GC) method can be summarized as follows: complex mixtures can be analyzed, only amounts in the nanogram-range or less are required, Kow values of isomers can be determined and the exact structure of compounds need not be known. Normalized GC retention data of persistent organic pollutants are readily available. The quality of the Kow values obtained by the GC method compares well with that for other Kow estimation methods. It depends mainly on the accuracy of the Kow data of the structurally correlated compounds used as standards for the correlation cohort. The Kow(GC) data for all 209 PCB congeners are given.