Reactive-site-centric chemoproteomics identifies a distinct class of deubiquitinase enzymes.

Activity-based probes (ABPs) are widely used to monitor the activity of enzyme families in biological systems. Inferring enzyme activity from probe reactivity requires that the probe reacts with the enzyme at its active site; however, probe-labeling sites are rarely verified. Here we present an enhanced chemoproteomic approach to evaluate the activity and probe reactivity of deubiquitinase enzymes, using bioorthogonally tagged ABPs and a sequential on-bead digestion protocol to enhance the identification of probe-labeling sites.

USP7 small-molecule inhibitors interfere with ubiquitin binding.

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation.

DUBs, the regulation of cell identity and disease.

The post-translational modification of proteins with ubiquitin represents a complex signalling system that co-ordinates essential cellular functions, including proteolysis, DNA repair, receptor signalling and cell communication. DUBs (deubiquitinases), the enzymes that disassemble ubiquitin chains and remove ubiquitin from proteins, are central to this system. Reflecting the complexity and versatility of ubiquitin signalling, DUB activity is controlled in multiple ways.

DNA recognition by a σ(54) transcriptional activator from Aquifex aeolicus.

Transcription initiation by bacterial σ(54)-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain (DBD). The structurally characterized DBDs from activators all belong to the Fis (factor for inversion stimulation) family of helix-turn-helix DNA-binding proteins.

Dual recruitment of Cdc48 (p97)-Ufd1-Npl4 ubiquitin-selective segregase by small ubiquitin-like modifier protein (SUMO) and ubiquitin in SUMO-targeted ubiquitin ligase-mediated genome stability functions.

Protein modification by SUMO and ubiquitin critically impacts genome stability via effectors that "read" their signals using SUMO interaction motifs or ubiquitin binding domains, respectively. A novel mixed SUMO and ubiquitin signal is generated by the SUMO-targeted ubiquitin ligase (STUbL), which ubiquitylates SUMO conjugates. Herein, we determine that the "ubiquitin-selective" segregase Cdc48-Ufd1-Npl4 also binds SUMO via a SUMO interaction motif in Ufd1 and can thus act as a selective receptor for STUbL targets.

SUMO-targeted ubiquitin ligase, Rad60, and Nse2 SUMO ligase suppress spontaneous Top1-mediated DNA damage and genome instability.

Through as yet undefined proteins and pathways, the SUMO-targeted ubiquitin ligase (STUbL) suppresses genomic instability by ubiquitinating SUMO conjugated proteins and driving their proteasomal destruction. Here, we identify a critical function for fission yeast STUbL in suppressing spontaneous and chemically induced topoisomerase I (Top1)-mediated DNA damage. Strikingly, cells with reduced STUbL activity are dependent on tyrosyl-DNA phosphodiesterase 1 (Tdp1).

Variability analysis of human plasma and cerebral spinal fluid reveals statistical significance of changes in mass spectrometry-based metabolomics data.

Analytical and biological variability are issues of central importance to human metabolomics studies. Here both types of variation are examined in human plasma and cerebrospinal fluid (CSF) using a global liquid chromatography/mass spectrometry (LC/MS) metabolomics strategy. The platform shows small analytical variation with a median coefficient of variation (CV) of 15-16% for both plasma and CSF sample matrixes when the integrated area of each peak in the mass spectra is considered.

Structure and regulatory mechanism of Aquifex aeolicus NtrC4: variability and evolution in bacterial transcriptional regulation.

Genetic changes lead gradually to altered protein function, making deduction of the molecular basis for activity from a sequence difficult. Comparative studies provide insights into the functional consequences of specific changes. Here we present structural and biochemical studies of NtrC4, a sigma-54 activator from Aquifex aeolicus, and compare it with NtrC1 (a paralog) and NtrC (a homolog from Salmonella enterica) to provide insight into how a substantial change in regulatory mechanism may have occurred.

Phosphatases, DNA damage checkpoints and checkpoint deactivation.

Cells have evolved intricate and specialized responses to DNA damage, central to which are the DNA damage checkpoints that arrest cell cycle progression and facilitate the repair process. Activation of these damage checkpoints relies heavily on the activity of Ser/Thr kinases, such as Chk1 and Chk2 (Saccharomyces cerevisiae Rad53), which are themselves activated by phosphorylation. Only more recently have we begun to understand how cells disengage the checkpoints to reenter the cell cycle.

Crystal structure of archaeal ribonuclease P protein aRpp29 from Archaeoglobus fulgidus.

The crystal structure of ribonuclease P protein aRpp29 from the sulfate-reducing hyperthermophile Archaeoglobus fulgidus was determined at 1.7 A resolution using X-ray diffraction methods. The central feature of this archaeal protein is a sheet of six antiparallel beta-strands twisted around a conserved hydrophobic core.