Small subcellular localization

SARA is a scaffolding protein that regulates the subcellular localization of inactivated R-Smads, potentially scaffolding the TGF-P receptor kinase to the Smad 2 substrate. Filamins are a family of actin polymerization proteins that also form scaffolds for a range of signaling proteins including SAP kinases such as MKK-4, small GTPases Rho and Ras, as well as Smad 2 and Smad 5. 

Fig. 9.1 Schematic diagram depicting drug transporters and their subcellular localization in the human small intestinal enterocyte (A), hepatocyte (B), and renal tubular cell (C).

Here we describe the construction and use of adenoviruses to express lucif-erase- or GFP-fusion proteins for high-throughput cell-based screens. We constructed adenoviruses that express a cyclin Bl-luciferase reporter protein or unmodified luciferase. This enabled us to screen for small molecules that specifically up-regulated levels of the cyclin B 1-luciferase fusion protein without affecting luciferase. A similar approach was used to construct adenoviruses that express cyclin A-GFP or cyclin Bl-GFP fusion proteins. These viruses were used in an imaging-based screen to identify small molecules that specifically upregulated these proteins or altered their subcellular localization. 

The score/penalty assigned to a match/mismatch is dependent on the likelihood that this match/mismatch occurred by chance alone. If an event occurs randomly with a high frequency the score will be small, while very rare events will receive very large scores. For example, a conservative amino-acid substitution will receive a very small penalty, while the introduction of a stop codon will be penalized heavily. A variety of scoring schemes have been generated based on factors such as the bio-physical character, evolutionary distance, and the subcellular localization of the sequences being compared. 

As many ER-retained mutant proteins are functional, rerouting them to their appropriate subcellular localization would restore the phenotype caused by their mislocalization. In some cases, it is estimated that only a small amount of functional protein (10-15%) is necessary to maintain health. Indeed, for some diseases a critical threshold seems to exist (7). Patients who express functional protein above this threshold have mild or no symptoms of the disease (7, 8), which suggests that even a modest increase in protein rerouting would improve the quality of life for many patients with these diseases. This finding provides the impetus for identifying correctors, even those with modest effects. 

Even without the use of additional reporters, small-molecule imaging provides a wealth of information that can inform and facilitate the triaging of potential lead candidates. Perhaps, the most critical information obtained from these microscopy studies is the subcellular localization (and therein cell permeability) and biodistribution of the small molecule additionally, the accumulated local concentration and half-life of the probe can be determined for in vivo models [28]. The use of a second reporter, that is, labeled antibody or fluorescent protein, can demonstrate target engagement within a biological environment... 

Fio. 3. Subcellular localization of endogenous NA and tritiated NA( H-NA) in rat vas deferens. After the administration of H-NA in vivo, the tBsue was homogenized and layered onto a sucrose density gradient for centrifugation. Appearance of the tube after centrifugation is indicated on the left. A hole was made in the bottom and the contents collected in five-drop fracticms. Alternate fractions were assayed for H-NA and for endogenous NA. Both n re distributed in the supernatant fluid and in a fraction of small vesicles just below the supernatant, which also contained microsomal particles. (Reproduced with permission frcnn Potter, L. T. and Axelrod, J., J. Pharmac. Exp. Ther., 142, 291-298, 1963.)... 

The intracellular localization of carboxylesterases is predominantly microsomal, the esterases being localized in the endoplasmic reticulum [73] [79] [93], They are either free in the lumen or loosely bound to the inner aspect of the membrane. The carboxylesterases in liver mitochondria are essentially identical to those of the microsomal fraction. In contrast, carboxylesterases of liver lysosomes are different, their isoelectric point being in the acidic range. Carboxylesterase activity is also found in the cytosolic fraction of liver and kidney. It has been suggested that cytosolic carboxylesterases are mere contaminants of the microsomal enzymes, but there is evidence that soluble esterases do not necessarily originate from the endoplasmic reticulum [94], In guinea pig liver, a specific cytosolic esterase has been identified that is capable of hydrolyzing acetylsalicylate and that differs from the microsomal enzyme. Also, microsomal and cytosolic enzymes have different electrophoretic properties [77]. Cytosolic and microsomal esterases in rat small intestinal mucosa are clearly different enzymes, since they hydrolyze rac-oxazepam acetate with opposite enantioselectivity [95], Consequently, studies of hydrolysis in hepatocytes reflect more closely the in vivo hepatic hydrolysis than subcellular fractions, since cytosolic and microsomal esterases can act in parallel. 

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