Systems-based design of bi-ligand inhibitors of oxidoreductases: filling the chemical proteomic toolbox.

Genomics-driven growth in the number of enzymes of unknown function has created a need for better strategies to characterize them. Since enzyme inhibitors have traditionally served this purpose, we present here an efficient systems-based inhibitor design strategy, enabled by bioinformatic and NMR structural developments. First, we parse the oxidoreductase gene family into structural subfamilies termed pharmacofamilies, which share pharmacophore features in their cofactor binding sites.

UBA domains mediate protein-protein interactions between two DNA damage-inducible proteins.

The Saccharomyces cerevisiae genes RAD23 and DDI1 were identified in a screen for multicopy suppressors of the temperature-sensitivity of a mutant allele of S. cerevisiae PDS1. Pds1 is a regulator of anaphase that needs to accumulate and then be degraded by the ubiquitin-proteasome pathway at the metaphase-anaphase transition for cells to progress normally through mitosis.

UBA domains of DNA damage-inducible proteins interact with ubiquitin.

Rad23 is a highly conserved protein involved in nucleotide excision repair (NER) that associates with the proteasome via its N-terminus. Its C-terminal ubiquitin-associated (UBA) domain is evolutionarily conserved from yeast to humans. However, the cellular function of UBA domains is not completely understood.

Dosage suppressors of pds1 implicate ubiquitin-associated domains in checkpoint control.

In budding yeast, anaphase initiation is controlled by ubiquitin-dependent degradation of Pds1p. Analysis of pds1 mutants implicated Pds1p in the DNA damage, spindle assembly, and S-phase checkpoints. Though some components of these pathways are known, others remain to be identified.

Microinjection of anti-p21 antibodies induces senescent Hs68 human fibroblasts to synthesize DNA but not to divide.

Replicative senescence is characterized by irreversible growth arrest and has been defined by four genetic complementation groups. One of these groups is associated with the predominance of underphosphorylated, growth-suppressive retinoblastoma tumor suppressor protein (pRb). Although certain members of the cyclin-dependent kinase (cdk)/cyclin family, some of which phosphorylate pRb, are underexpressed in senescent cells, others are expressed but inactive.

Cyclin-dependent kinase and Cks/Suc1 interact with the proteasome in yeast to control proteolysis of M-phase targets.

Cell cycle-specific proteolysis is critical for proper execution of mitosis in all eukaryotes. Ubiquitination and subsequent proteolysis of the mitotic regulators Clb2 and Pds1 depend on the cyclosome/APC and the 26S proteasome. We report here that components of the cell cycle machinery in yeast, specifically the cell cycle regulatory cyclin-dependent kinase Cdc28 and a conserved associated protein Cks1/Suc1, interact genetically, physically, and functionally with components of the 26S proteasome.

Elevated cyclins and cyclin-dependent kinase activity in the rhabdomyosarcoma cell line RD.

An important early event in the differentiation of skeletal muscle cells is exit from the cell cycle, after which full expression of the muscle phenotype occurs. Rhabdomyosarcoma (RMS), a tumor of skeletal muscle origin, expresses a number of muscle-specific proteins, including MyoD; however, these cells fail to arrest or differentiate when cultured in differentiation medium (DM). To determine the basis for the failure of RMS cells to differentiate or arrest, we studied the molecular response of the embryonal RMS cell line, RD, to culture in DM.

Human peroxisomal targeting signal-1 receptor restores peroxisomal protein import in cells from patients with fatal peroxisomal disorders.

Two peroxisomal targeting signals, PTS1 and PTS2, are involved in the import of proteins into the peroxisome matrix. Human patients with fatal generalized peroxisomal deficiency disorders fall into at least nine genetic complementation groups. Cells from many of these patients are deficient in the import of PTS1-containing proteins, but the causes of the protein-import defect in these patients are unknown.

Complementation of fragments of triosephosphate isomerase defined by exon boundaries.

Chicken triosephosphate isomerase (TIM) has been fragmented by inserting single "splits" at three separate exon/exon boundaries, and the complexes have been assayed for catalytic activity. In vivo studies show that the expression of both portions of each of the three different split genes complements the TIM deficiency of Escherichia coli strain DF502 when grown on selective media. The expression of only one fragment of each split gene does not complement the TIM-minus genotype.