SCRATCH: a protein structure and structural feature prediction server

SCRATCH is a server for predicting protein tertiary structure and structural features. The SCRATCH software suite includes predictors for secondary structure, relative solvent accessibility, disordered regions, domains, disulfide bridges, single mutation stability, residue contacts versus average, individual residue contacts and tertiary structure. The user simply provides an amino acid sequence and selects the desired predictions, then submits to the server. Results are emailed to the user. The server is available at

Comprehensive proteomic profiling of outer membrane vesicles from Campylobacter jejuni

Gram-negative bacteria constitutively release outer membrane vesicles (OMVs) during cell growth that play significant roles in bacterial survival, virulence and pathogenesis. In this study, comprehensive proteomic analysis of OMVs from a human gastrointestinal pathogen Campylobacter jejuni NCTC11168 was performed using high-resolution mass spectrometry. The OMVs of C. jejuni NCTC11168 were isolated from culture supernatants then characterized using electron microscopy and dynamic light scattering revealing spherical OMVs of an average diameter of 50 nm. We then identified 134 vesicular proteins using high-resolution LTQ-Orbitrap mass spectrometry. Subsequent functional analysis of the genes revealed the relationships of the vesicular proteins. Furthermore, known N-glycoproteins were identified from the list of the vesicular proteome, implying the potential role of the OMVs as a delivery means for biologically relevant bacterial glycoproteins. These results enabled us to elucidate the overall proteome profile of pathogenic bacterium C. jejuni and to speculate on the function of OMVs in bacterial infections and communication

Ribosomal proteins produced in excess are degraded by the ubiquitin-proteasome system

Ribosome assembly is an essential process that consumes prodigious quantities of cellular resources. Ribosomal proteins cannot be overproduced in Saccharomyces cerevisiae because the excess proteins are rapidly degraded. However, the responsible quality-control (QC) mechanisms remain poorly characterized. Here, we demonstrate that overexpression of multiple proteins of the small and large yeast ribosomal subunits is suppressed. Rpl26 overexpressed from a plasmid can be detected in the nucleolus and nucleoplasm, but it largely fails to assemble into ribosomes and is rapidly degraded. However, if the endogenous RPL26 loci are deleted, plasmid-encoded Rpl26 assembles into ribosomes and localizes to the cytosol. Chemical and genetic perturbation studies indicate that overexpressed ribosomal proteins are degraded by the ubiquitin-proteasome system, and not by autophagy. Inhibition of the proteasome led to accumulation of multiple endogenous ribosomal proteins in insoluble aggregates, consistent with the operation of this QC mechanism in the absence of ribosomal protein overexpression. Our studies reveal that ribosomal proteins that fail to assemble into ribosomes are rapidly distinguished from their assembled counterparts and are ubiquitinated and degraded within the nuclear compartment

PIKES Analysis Reveals Response to Degraders and Key Regulatory Mechanisms of the CRL4 Network

Co-opting Cullin4 RING ubiquitin ligases (CRL4s) to inducibly degrade pathogenic proteins is emerging as a promising therapeutic strategy. Despite intense efforts to rationally design degrader molecules that co-opt CRL4s, much about the organization and regulation of these ligases remains elusive. Here, we establish protein interaction kinetics and estimation of stoichiometries (PIKES) analysis, a systematic proteomic profiling platform that integrates cellular engineering, affinity purification, chemical stabilization, and quantitative mass spectrometry to investigate the dynamics of interchangeable multiprotein complexes. Using PIKES, we show that ligase assemblies of Cullin4 with individual substrate receptors differ in abundance by up to 200-fold and that Cand1/2 act as substrate receptor exchange factors. Furthermore, degrader molecules can induce the assembly of their cognate CRL4, and higher expression of the associated substrate receptor enhances degrader potency. Beyond the CRL4 network, we show how PIKES can reveal systems level biochemistry for cellular protein networks important to drug development

Single subunit degradation of WIZ, a lenalidomide- and pomalidomide dependent substrate of E3 ubiquitin ligase CRL4^(CRBN)

Immunomodulators (IMiDs) are an effective class of drugs used to treat blood cancers. IMiDs are believed to work by recruiting protein targets containing a β-hairpin motif for ubiquitination by E3 ubiquitin ligase complexes composed of cereblon (CRBN), Cullin-4a (CUL4a), DNA Damage Binding protein-1 (DDB1), and Ring Box-1 (RBX1). The ubiquitinated protein is subsequently degraded by the proteasome. By characterizing the repertoire of proteins that show an increased physical association with CRBN after IMiD treatment, we identified a novel IMiD substrate, Widely Interspaced Zinc Finger Motifs (WIZ). WIZ contains a C2H2 zinc finger domain, like several other substrates that were previously characterized. We demonstrate that IMiDs stabilize physical association of WIZ with CRBN, deplete WIZ steady state protein levels in a way that is dependent on E3 ligase activity, and enhance the rate of its degradation. Notably, proteins that assemble with WIZ are co-recruited to CRBN by IMiDs but are not degraded, illustrating the potential of targeted protein degradation to eliminate individual subunits of a protein complex. These findings suggest that systematic characterization of the full repertoire of proteins that are targeted for degradation by IMiD compounds will be required to better understand their biological effects

Composition and Regulation of the Cellular Repertoire of SCF Ubiquitin Ligases

SCF (Skp1-Cullin-F-box) ubiquitin ligases comprise several dozen modular enzymes that have diverse roles in biological regulation. SCF enzymes share a common catalytic core containing Cul1⋅Rbx1, which is directed toward different substrates by a variable substrate receptor (SR) module comprising 1 of 69 F-box proteins bound to Skp1. Despite the broad cellular impact of SCF enzymes, important questions remain about the architecture and regulation of the SCF repertoire, including whether SRs compete for Cul1 and, if so, how this competition is managed. Here, we devise methods that preserve the in vivo assemblages of SCF complexes and apply quantitative mass spectrometry to perform a census of these complexes (the “SCFome”) in various states. We show that Nedd8 conjugation and the SR exchange factor Cand1 have a profound effect on shaping the SCFome. Together, these factors enable rapid remodeling of SCF complexes to promote biased assembly of SR modules bound to substrate

Oxidation of p53 through DNA Charge Transport Involves a Network of Disulfides within the DNA-Binding Domain

Transcription factor p53 plays a critical role in the cellular response to stress stimuli. We have seen that p53 dissociates selectively from various promoter sites as a result of oxidation at long-range through DNA-mediated charge transport (CT). Here, we examine this chemical oxidation and determine the residues in p53 that are essential for oxidative dissociation, focusing on the network of cysteine residues adjacent to the DNA-binding site. Of the eight mutants studied, only the C275S mutation shows decreased affinity for the Gadd45 promoter site. However, both mutations C275S and C277S result in substantial attenuation of oxidative dissociation, with C275S causing the most severe attenuation. Differential thiol labeling was used to determine the oxidation states of cysteine residues within p53 after DNA-mediated oxidation. Reduced cysteines were iodoacetamide-labeled, whereas oxidized cysteines participating in disulfide bonds were ^(13)C_2D_2-iodoacetamide-labeled. Intensities of respective iodoacetamide-modified peptide fragments were analyzed by mass spectrometry. A distinct shift in peptide labeling toward ^(13)C_2D_2-iodoacetamide-labeled cysteines is observed in oxidized samples, confirming that chemical oxidation of p53 occurs at long range. All observable cysteine residues trend toward the heavy label under conditions of DNA CT, indicating the formation of multiple disulfide bonds among the cysteine network. On the basis of these data, it is proposed that disulfide formation involving C275 is critical for inducing oxidative dissociation of p53 from DNA

SutA is a bacterial transcription factor expressed during slow growth in Pseudomonas aeruginosa

Microbial quiescence and slow growth are ubiquitous physiological states, but their study is complicated by low levels of metabolic activity. To address this issue, we used a time-selective proteome-labeling method [bioorthogonal noncanonical amino acid tagging (BONCAT)] to identify proteins synthesized preferentially, but at extremely low rates, under anaerobic survival conditions by the opportunistic pathogen Pseudomonas aeruginosa. One of these proteins is a transcriptional regulator that has no homology to any characterized protein domains and is posttranscriptionally up-regulated during survival and slow growth. This small, acidic protein associates with RNA polymerase, and chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing suggests that the protein associates with genomic DNA through this interaction. ChIP signal is found both in promoter regions and throughout the coding sequences of many genes and is particularly enriched at ribosomal protein genes and in the promoter regions of rRNA genes. Deletion of the gene encoding this protein affects expression of these and many other genes and impacts biofilm formation, secondary metabolite production, and fitness in fluctuating conditions. On the basis of these observations, we have designated the protein SutA (survival under transitions A)