Advances in directed protein evolution by recursive genetic recombination: applications to therapeutic proteins.

Recent developments in directed evolution technologies combined with innovations in robotics and screening methods have revolutionized protein engineering. These methods are being applied broadly to many fields of biotechnology, including chemical engineering, agriculture and human therapeutics. More specifically, DNA shuffling and other methods of genetic recombination and mutation have resulted in the improvement of proteins of therapeutic interest. Optimizing genetic diversity and fitness through iterative directed evolution will accelerate improvements in engineered protein therapeutics.

Ubc9 interacts with a nuclear localization signal and mediates nuclear localization of the paired-like homeobox protein Vsx-1 independent of SUMO-1 modification.

Vsx-1 is a paired-like:CVC homeobox gene whose expression is linked to bipolar cell differentiation during zebrafish retinogenesis. We used a yeast two-hybrid screen to identify proteins interacting with Vsx-1 and isolated Ubc9, an enzyme that conjugates the small ubiquitin-like modifier SUMO-1. Despite its interaction with Ubc9, we show that Vsx-1 is not a substrate for SUMO-1 in COS-7 cells or in vitro. When a yeast two-hybrid assay is used, deletion analysis of the interacting domain on Vsx-1 shows that Ubc9 binds to a nuclear localization signal (NLS) at the NH(2) terminus of the homeodomain. In SW13 cells, Vsx-1 localizes to the nucleus and is excluded from nucleoli. Deletion of the NLS disrupts this nuclear localization, resulting in a diffuse cytoplasmic distribution of Vsx-1. In SW13 AK1 cells that express low levels of endogenous Ubc9, Vsx-1 accumulates in a perinuclear ring and colocalizes with an endoplasmic reticulum marker. However, NLS-tagged STAT1 protein exhibits normal nuclear localization in both SW13 and SW13 AK1 cells, suggesting that nuclear import is not globally disrupted. Cotransfection of Vsx-1 with Ubc9 restores Vsx-1 nuclear localization in SW3 AK1 cells and demonstrates that Ubc9 is required for the nuclear localization of Vsx-1. Ubc9 continues to restore nuclear localization even after a C93S active site mutation has eliminated its SUMO-1-conjugating ability. These results suggest that Ubc9 mediates the nuclear localization of Vsx-1, and possibly other proteins, through a nonenzymatic mechanism that is independent of SUMO-1 conjugation.

Ubiquitination and degradation of the zebrafish paired-like homeobox protein VSX-1.

Vsx-1 is a paired-like : CVC homeobox protein dynamically expressed during zebrafish development. Previous results indicate that Vsx-1 influences bipolar cell differentiation and maintenance of these cells in the adult retina. To understand the developmental regulation of this transcription factor, we investigated ubiquitination as a possible posttranslational mechanism. In vitro, Vsx-1 was conjugated with multiple ubiquitin moieties. Proteasome inhibitors and added ubiquitin increased the accumulation of Vsx-1-ubiquitin(n) complexes and stabilized unmodified Vsx-1. Also, in transiently transfected COS-7 cells, Vsx-1 is ubiquitinated, and pulse-chase experiments show that Vsx-1 proteolysis occurs. Vsx-1 proteins with C-terminal deletions retained the capacity for initial modification by ubiquitin but lost the capacity for efficient chain elongation. These results show that Vsx-1 is a substrate of the ubiquitin/proteasome pathway and suggest that C-terminal sequences of Vsx-1 are critical for ubiquitin chain elongation. In addition, our findings suggest that ubiquitin-dependent proteolysis regulates Vsx-1 during zebrafish retinal development.

Cloning of zebrafish vsx1: expression of a paired-like homeobox gene during CNS development.

vsx1 is a homeobox gene encoding a paired-type homeodomain and a CVC domain that was originally cloned from an adult goldfish retinal library. We previously reported the spatiotemporal expression pattern of vsx1 in the adult and developing retina of zebrafish and goldfish, and we suggested that vsx1 plays a role in determining the cell fate and maintenance of retinal interneurons. Other related genes encoding a CVC domain, such as vsx2 (alx) and chx10, are expressed both within and outside the retina during development. In this study, we report the cloning of zebrafish vsx1 and its developmental expression in both retinal and nonretinal regions of the CNS in zebrafish embryos. vsx1 expression was detected in a subset of hindbrain and spinal cord neurons before it was expressed in the retina. At about the same time that retinal expression began, the level of vsx1 was decreased in the spinal cord. The expression of vsx1 was progressively restricted, and eventually it was detected only in the inner nuclear layer (INL) of the developing retina. The combined expression patterns of teleost vsx1 and vsx2 (alx) during early zebrafish development encompasses the expression pattern observed for murine Chx10, and indicates a partitioning of function for CVC genes in lower vertebrates.

On the mechanism of lithium-induced renal tubular acidosis.

The effect of lithium chloride administration on urinary acidification was studied in dogs. Lithium-treated dogs developed hyperchloremic metabolic acidosis with alkaline urine. Bicarbonate loading resulted in a normal increase in urinary Pco2 in normal dogs but failed to produce the same response in lithium-treated dogs. The bicarbonate titration curve of lithium-treated dogs revealed a small leak of bicarbonate at low plasma levels of bicarbonate; at high plasma levels bicarbonate reabsorption was significantly higher in lithium-treated dogs. This pattern of bicarbonate reabsorption is identical to that described in classic distal renal tubular acidosis. Sodium sulfate administration resulted in a normal urinary acidification ilithium-treated dogs. It is possible that lithium administration induces distal renal tubular acidosis by allowing excessive back-diffusion of acid. This excessive back-diffusion of acid would result in a low urinary Pco2 during bicarbonate loading. Sodium sulfate administration, by increasing the negative intratubular potential, would restrict back-diffusion of hydrogen ion and thereby result in a normal acidification in lithium-treated dogs. We previously demonstrated that postureteral obstruction of the kidney fails to increase urinary Pco2 during bicarbonate loading and to lower urinary pH with sodium sulfate. It is possible that a low urinary Pco2 during HCO3 loading can occur as a consequence of either diminished hydrogen ion secretion (postobstructed kidney) or excessive back-diffusion of acid (lithium administration). Further studies are indicated to determine whether both mechanisms may be found in patients with distal renal tubular acidosis.