Ubiquitin ligases are well suited to regulate molecular networks that operate on a post-translational timescale. The F-box family of proteins - which are the substrate-recognition components of the Skp1-Cul1-F-box-protein (SCF) ubiquitin ligase - are important players in many mammalian functions. Here we explore a unifying and structurally detailed view of SCF-mediated proteolytic control of cellular processes that has been revealed by recent studies.

The SCF ubiquitin ligase: insights into a molecular machine

In the year 2003 there was a 17% increase in the number of publications citing work performed using optical biosensor technology compared with the previous year. We collated the 962 total papers for 2003, identified the geographical regions where the work was performed, highlighted the instrument types on which it was carried out, and segregated the papers by biological system. In this overview, we spotlight 13 papers that should be on everyone's 'must read' list for 2003 and provide examples of how to identify and interpret high-quality biosensor data. Although we still find that the literature is replete with poorly performed experiments, over-interpreted results and a general lack of understanding of data analysis, we are optimistic that these shortcomings will be addressed as biosensor technology continues to mature.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/jmr.753

Survey of the year 2003 commercial optical biosensor literature

A hitchhiker's guide to the cullin ubiquitin ligases: SCF and its kin.

Lanthanides have recently found applications in different fields of biomolecular and medical research. Luminescent lanthanide chelates have created interest mainly due to their unique luminescent properties, such as their long Stokes’ shift and exceptional decay times allowing efficient temporal discrimination of background interferences in the assays, such as immunoassays. Recently, new organometallic complexes have been developed giving opportunities to novel applications, in heterogeneous and homogeneous immunoassays, DNA hybridization assays, high-throughput screening as well as in imaging. In addition, encapsulating the chelates into suitable matrix in beads enables the use of new members of lanthanides extending the emission wavelength to micrometer range and decays from a few microseconds to milliseconds. As the luminescence is derived from complicated intrachelate energy transfer, it also gives novel opportunities to exploit these levels in different types of energy transfer based applications. This review gives a short overview of recent development of lanthanide chelate-labels and discusses in more details of energy levels and their exploitation in new assay formats.

Progress in lanthanides as luminescent probes.

Time-resolved luminescence bioassays based on lanthanide-luminescence probes have become common, useful tools in clinical diagnostics and bioanalytical chemistry. The unique spectral and temporal properties of lanthanide-luminescence probes, such as very long luminescence lifetime, large Stokes Shift and sharp emission profile, make them an ideal choice for use in time-resolved luminescence bioassays to discriminate background noises effectively. This review gives an overview of recent developments in lanthanide-luminescence probes and their applications in time-resolved luminescence bioassays.

Lanthanide-based luminescence probes and time-resolved luminescence bioassays

IL-2-inducible T cell kinase plays an essential role in T cell receptor signaling and is considered a drug target for the treatment of Th2-mediated inflammatory diseases. By applying high-throughput protein engineering and crystallization, we have determined the X-ray crystal structures of IL-2-inducible T cell kinase in complex with its selective inhibitor BMS-509744 and the broad-spectrum kinase inhibitors sunitinib and RO5191614. Sunitinib uniquely stabilizes IL-2-inducible T cell kinase in the helix C-in conformation by inducing side chain conformational changes in the ATP-binding site. This preference of sunitinib to bind to an active kinase conformation is reflective of its broad-spectrum kinase activity. BMS-509744 uniquely stabilizes the activation loop in a substrate-blocking inactive conformation, indicating that structural changes described for Src family kinases are also involved in the regulation of IL-2-inducible T cell kinase activity. The observed BMS-509744 binding mode allows rationalization of structure–activity relationships reported for this inhibitor class and facilitates further structure-based drug design. Sequence-based analysis of this binding mode provides guidance for the rational design of inhibitor selectivity.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1747-0285.2010.00993.x

Crystal structures of IL-2-inducible T cell kinase complexed with inhibitors: insights into rational drug design and activity regulation.

The p27(Kip1) protein is a potent cyclin-dependent kinase inhibitor, the level of which is decreased in many common human cancers as a result of enhanced ubiquitin-dependent degradation. The multiprotein complex SCF(Skp2) has been identified as the ubiquitin ligase that targets p27, but the functional interactions within this complex are not well understood. One component, the F-box protein Skp2, binds p27 when the latter is phosphorylated on Thr(187), thus providing substrate specificity for the ligase. Recently, we and others have shown that the small cell cycle regulatory protein Cks1 plays a critical role in p27 ubiquitination by increasing the binding affinity of Skp2 for p27. Here we report the development of a homogeneous time-resolved fluorescence assay that allows the quantification of the molecular interactions between human recombinant Skp2, Cks1 and a p27-derived peptide phosphorylated on Thr(187). Using this assay, we have determined the dissociation constant of the Skp2-Cks1 complex (K(d) 140 +/- 14 nM) and have shown that Skp2 binds phosphorylated p27 peptide with high affinity only in the presence of Cks1 (K(d) 37 +/- 2 nM). Cks1 does not bind directly to the p27 phosphopeptide or to Skp1, which confirms its suggested role as an allosteric effector of Skp2.

Wei Chu

Papers

Crystal structures of IL-2-inducible T cell kinase complexed with inhibitors: insights into rational drug design and activity regulation.

Protein-protein interactions involved in the recognition of p27 by E3 ubiquitin ligase.