Cover story

Angiogenesis, the process of blood vessel growth, is hyperactivated in certain cancers. Thus, anti-angiogenic compounds, which may regulate tumor growth by restricting blood supply to malignant tissues, show great promise for cancer chemotherapy. Chan and co-workers have now developed a regenerative angiogenesis assay in the tailfin of adult zebrafish. They demonstrated that PTK787, a kinase inhibitor, could regulate angiogenesis in the adult caudal fin and that vascular endothelial growth factor receptor (VEGFR) signaling was required. Using zebrafish mutants, the authors were able to show that regenerative growth of bone and tissue was unaffected by chemical inhibition of angiogenesis. This angiogenesis assay, along with the chemical and genetic approach used by Chan and co-workers, provides a method for the rapid identification of anti-angiogenic lead compounds and a new route to discover genes and molecular pathways involved in angiogenesis. [Articles, p. 265 ; News & Views, p. 228 ] TLS

Functional proteomics of metalloproteases

With about 100 metalloproteases (MPs) in the human genome, it has been a challenge to characterize them individually. Also, as their activities are usually modified post-translationally, they have been difficult to monitor through proteomics-based strategies. Sieber, Cravatt and colleagues have now combined active site probing with proteome-based profiling to generate a library of new probes against a subset of MPs. A subgroup of four probes collectively targeted the vast majority of enzymes labeled by the library and allowed the authors to simultaneously monitor MPs having various catalytic mechanisms. Using these optimal probes, they were able to find differences in MP activities between invasive and noninvasive breast carcinomas and among melanoma cell lines. In addition, they found that they did indeed probe several MPs (including two integral-membrane MPs) that had not previously been accessible with activity-based probes. [Articles, p. 274 ; News & Views, p. 229 ] MB

Oxidative stress on the brain

Lewy body dementia and Parkinson disease are neurodegenerative diseases that are associated with the aggregation of α-synuclein into amyloid fibrils. Oxidative stress due to endogenous metabolism in dopaminergic neurons has been linked to enhanced neurodegeneration. Kelly and co-workers now suggest that cholesterol oxidative metabolism may provide the molecular link between oxidative stress and amyloid fibrilization in Lewy body disease. Cholesterol and α-synuclein are closely associated in neuronal membranes. The authors showed that oxidized cholesterol metabolites accumulated in the brains of individuals with Lewy body disease. The cholesterol aldehydes were also detected in cells showing oxidative stress and enhanced concentrations of α-synuclein. Using biophysical methods, the authors further showed that oxidized cholesterol metabolites induced α-synuclein fibrilization in vitro. The study highlights a potential cycle involving oxidative stress, cholesterol modification and α-synuclein aggregation that may contribute to Lewy body dementia. [Letters, p. 249 ] TLS

Ribosome form defines function

To help coordinate ribosomal activity, the 50S and 30S ribosomal subunits communicate with each other through an array of intersubunit bridges at their binding interface. Most of the large subunit interface consists of RNA-RNA interactions between the 16S rRNA of the 30S subunit and the 23S rRNA of the 50S subunit. Rackham, Chin and colleagues have generated libraries of mutations in the 16S rRNA and selected for those that could form functional ribosomes with wild-type translation activity. Mutations within many of the bridges were tolerated, suggesting that the informational content for the majority of bridges is low. From the less-tolerated mutations, the authors were able to define several functionally important molecular surfaces or “hotspots” that are disproportionately important for ribosome function. [Letters, p. 254 ; News & Views, p. 231 ] MB

Hydrogens seen clearly

Hydrogen bonding and proton transfer play key roles in enzymatic reactions. Recent advances have allowed atomic resolution crystallography to be used to identify the precise position of hydrogen atoms in enzyme structures. The enzymatic activity of cholesterol oxidase, which uses a flavin adenine dinucleotide cofactor to oxidize and isomerize 3β-hydroxysteroids, is pH sensitive. The kcat of the enzyme is independent of pH between pH 4.5 and 7.5 but is reduced by more than 1,000-fold above pH 7.5. Vrielink and colleagues have obtained five atomic-resolution crystallographic 'snapshots' of cholesterol oxidase over a pH range of 4.5 to 9.0. Above pH 7.5 they observed the anionic oxidized form of flavin adenine dinucleotide and the negatively charged imidazolate form of a key active site histidine. These observations now provide a molecular basis for understanding the effect of pH on cholesterol oxidase activity. [Letters, p. 259 ] JK

Reaction vessels in cell biology

To achieve and maintain a proper fold, a protein must adapt to the various compartments as it travels throughout the cell. Each of these cellular domains, including the ER, lysosomes, and even the lipid bilayer and the membrane-aqueous solvent interface, represents a different chemical environment. In a Commentary, Kelly and Balch discuss the connections among chemical biology, protein folding and compartmentalization in terms of distinct reaction vessels. These vessels are diverse and numerous, containing chaperones and small molecules that contribute to the protein-folding landscape by modulating the energetics of protein conformation. [Commentary, p. 224 ] MB

In this Issue written by Mirella Bucci, Joanne Kotz and Terry L. Sheppard