Biomedical subjects

Balaji M Rao

Publications and source records attributed to Balaji M Rao.

4 recordsLinked to original sources

PubMed2005

High-affinity CD25-binding IL-2 mutants potently stimulate persistent T cell growth.

We have used directed evolution to construct IL-2 mutants that bind the IL-2 alpha receptor subunit (IL-2Ralpha, CD25) with affinities comparable to that of the IL-15-IL-15 alpha receptor subunit (IL-15Ralpha) interaction. T cells proliferate for up to 6 days following a 30 minute incubation with these IL-2 mutants, which may lead to potential applications for cancer and viral immunotherapy. Several alternative mechanisms have been proposed to explain the contrasting effects of IL-2 and IL-15 on T cell proliferation and death. These IL-2 mutants exhibit T cell growth response-receptor occupancy curves indistinguishable from that for IL-15, suggesting that much of the difference between wild-type IL-2 and IL-15 effects arises simply from their 1000-fold differing affinities for their private alpha receptor subunits.

Amino Acid Substitution↗

PubMed2005

Integrating cell-level kinetic modeling into the design of engineered protein therapeutics.

Functional genomics and proteomics are identifying many potential drug targets for novel therapeutic proteins, and both rational and combinatorial protein engineering methods are available for creating drug candidates. A central challenge is the definition of the most appropriate design criteria, which will benefit critically from computational kinetic models that incorporate integration from the molecular level to the whole systems level. Interpretation of these processes will require mathematical models that are refined in combination with relevant data derived from quantitative assays, to correctly set biophysical objectives for protein design.

Cell Physiological Phenomena↗

PubMed2004

Identification of cytoskeletal [14C]carboplatin-binding proteins reveals reduced expression and disorganization of actin and filamin in cisplatin-resistant cell lines.

Cisplatin resistant (CP-r) cells often show decreased uptake of cisplatin in association with reduced cell surface proteins and decreased endocytosis. In this report, two major [14C]carboplatin-binding proteins were identified as filamin and actin by photoaffinity labeling and mass spectrometry. Decreased expression of these two proteins was found in two different human CP-r cell lines (KB-CP20 and 7404-CP20), in comparison with their parental cell lines (KB-3-1 and BEL-7404), respectively. Disorganization of beta-actin and filamin 250 and 90 was also detected in these CP-r cells by confocal microscopy. Transfection of a wild-type actin-enhanced green fluorescent protein (EGFP) expression vector into 7404-CP20 cells resulted in a nonfilamentous actin-EGFP distribution compared with a normal distribution in the cisplatin-sensitive BEL-7404 cells, suggesting that cytoskeletal organization is disturbed in the CP-r cells. The identification of actin and filamin as [14C]carboplatin-binding proteins and decreased expression and disorganization of several cytoskeletal proteins in CP-r cells provide a molecular and cellular basis for the known defect in endocytosis in these cells.

Actins↗

PubMed2003

Interleukin-2 mutants with enhanced alpha-receptor subunit binding affinity.

Stimulation of T-cells by IL-2 has been exploited for treatment of metastatic renal carcinoma and melanoma. However, a narrow therapeutic window delimited by negligible stimulation of T-cells at low picomolar concentrations and undesirable stimulation of NK cells at nanomolar concentrations hampers IL-2-based therapies. We hypothesized that increasing the affinity of IL-2 for IL-2Ralpha may create a class of IL-2 mutants with increased biological potency as compared with wild-type IL-2. Towards this end, we have screened libraries of mutated IL-2 displayed on the surface of yeast and isolated mutants with a 15-30-fold improved affinity for the IL-2Ralpha subunit. These mutants do not exhibit appreciably altered bioactivity at 0.5-5 pM in steady-state bioassays, concentrations well below the IL-2Ralpha equilibrium binding constant for both the mutant and wild-type IL-2. A mutant was serendipitously identified that exhibited somewhat improved potency, perhaps via altered endocytic trafficking mechanisms described previously.

Cell Division↗