The proteasome is the degradation machine at the center of the ubiquitin-proteasome system and controls the concentrations of many proteins in eukaryotes. It is highly processive so that substrates are degraded completely into small peptides, avoiding the formation of potentially toxic fragments. Nonetheless, some proteins are incompletely degraded, indicating the existence of factors that influence proteasomal processivity. We have quantified proteasomal processivity and determined the underlying rates of substrate degradation and release. We find that processivity increases with species complexity over a 5-fold range between yeast and mammalian proteasome, and the effect is due to slower but more persistent degradation by proteasomes from more complex organisms. A sequence stretch that has been implicated in causing incomplete degradation, the glycine-rich region of the NFκB subunit p105, reduces the proteasome’s ability to unfold its substrate, and polyglutamine repeats such as found in Huntington’s disease reduce the processivity of the proteasome in a length-dependent manner.

The hyperthermophilic bacterium Thermotoga maritima has shared many genes with archaea through horizontal gene transfer. Several of these encode putative oligopeptide ATP binding cassette (ABC) transporters. We sought to test the hypothesis that these transporters actually transport sugars by measuring the substrate affinities of their encoded substrate-binding proteins (SBPs). This information will increase our understanding of the selective pressures that allowed this organism to retain these archaeal homologs. By measuring changes in intrinsic fluorescence of these SBPs in response to exposure to various sugars, we found that five of the eight proteins examined bind to sugars. We could not identify the ligands of the SBPs TM0460, TM1150, and TM1199. The ligands for the archaeal SBPs are TM0031 (BglE), the ␤-glucosides cellobiose and laminaribiose; TM0071 (XloE), xylobiose and xylotriose; TM0300 (GloE), large glucose oligosaccharides represented by xyloglucans; TM1223 (ManE), ␤-1,4-mannobiose; and TM1226 (ManD), ␤-1,4-mannobiose, ␤-1,4-mannotriose, ␤-1,4-mannotetraose, ␤-1,4-galactosyl mannobiose, and cellobiose. For comparison, seven bacterial putative sugar-binding proteins were examined and ligands for three (TM0595, TM0810, and TM1855) were not identified. The ligands for these bacterial SBPs are TM0114 (XylE), xylose; TM0418 (InoE), myo-inositol; TM0432 (AguE), ␣-1,4-digalactouronic acid; and TM0958 (RbsB), ribose. We found that T. maritima does not grow on several complex polypeptide mixtures as sole sources of carbon and nitrogen, so it is unlikely that these archaeal ABC transporters are used primarily for oligopeptide transport. Since these SBPs bind oligosaccharides with micromolar to nanomolar affinities, we propose that they are used primarily for oligosaccharide transport.

Metastatic tumor cells colonize the pre-metastatic niche, which is a complex microenvironment consisting partially of extracellular matrix (ECM) proteins. We sought to identify and validate novel contributors to tumor cell colonization using ECM coated poly(ε-caprolactone) (PCL) scaffolds as mimics of the pre-metastatic niche. Utilizing orthotopic breast cancer mouse models, fibronectin and collagen IV-coated scaffolds implanted in the subcutaneous space captured colonizing tumor cells, showing a greater than 2-fold increase in tumor cell accumulation at the implant site compared to uncoated scaffolds. As a strategy to identify additional ECM colonization contributors, decellularized matrix (DCM) from lungs and livers containing metastatic tumors were characterized. In vitro, metastatic cell adhesion was increased on DCM coatings from diseased organs relative to healthy DCM. Furthermore, in vivo implantations of diseased DCM-coated scaffolds had increased tumor cell colonization relative to healthy DCM coatings. Mass-spectrometry proteomics was performed on healthy and diseased DCM to identify candidates associated with colonization. Myeloperoxidase was identified as abundantly present in diseased organs and validated as a contributor to colonization using myeloperoxidase-coated scaffold implants. This work identified novel ECM proteins associated with colonization using decellularization and proteomics techniques and validated candidates using a scaffold to mimic the pre-metastatic niche.

In this study, high-affinity maltose-and glucose-binding activities in cell-free extracts of Thermotoga maritima were detected ; these activities were distinct and specific. At the gross level, the expression of binding-protein activities was repressed by growth of T. maritima in the presence of the cognate sugar. Growth of the organism in the presence of maltose reduced maltose-binding activity but not glucose-binding activity, while growth in the presence of glucose reduced glucose-binding activity but not maltose-binding activity. In competition assays, these binding activities showed distinct patterns of substrate specificity : whereas the maltose-binding activity showed specificity for α-linked glucosides, the glucose-binding activity showed a broader specificity. All maltose-and glucose-binding activity was found in the supernatant retrieved following centrifugation (100 000 g) of the cell-free extracts prepared by French-pressure-cell treatment ; no activity was found in an octyl-glucoside-treated extract of the membrane fraction. The maltosebinding-protein activity was recovered from the periplasmic fraction by selective release of the periplasmic contents of T. maritima cells using a newly developed freeze-thaw procedure. Annotation of the complete genome sequence of T. maritima suggests that there may be at least two maltosebinding proteins, MalE1 and MalE2, encoded in the genome. The maltosebinding activity corresponded to a protein of 43 kDa, which was consistent in size with either of the putative proteins. These data demonstrate that the hyperthermophilic bacterium T. maritima possesses separate maltose-and glucose-binding-protein activities that are freely soluble in its periplasm, in contrast to the membrane-bound sugar-binding proteins found in archaeal hyperthermophiles.

Significance: Bioactivity and receptor-ligand affinity assays developed herein provide a platform to discover quorum-sensing inhibitors.

Protein ubiquitination is mediated sequentially by ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3. Uba1 was thought to be the only E1 until the recent identification of Uba6. To differentiate the biological functions of Uba1 and Uba6, we applied an orthogonal ubiquitin transfer (OUT) technology to profile their ubiquitination targets in mammalian cells. By expressing pairs of an engineered ubiquitin and engineered Uba1 or Uba6 that were generated for exclusive interactions, we identified 697 potential Uba6 targets and 527 potential Uba1 targets with 258 overlaps. Bioinformatics analysis reveals substantial differences in pathways involving Uba1- and Uba6-specific targets. We demonstrate that polyubiquitination and proteasomal degradation of ezrin and CUGBP1 require Uba6, but not Uba1, and that Uba6 is involved in the control of ezrin localization and epithelial morphogenesis. These data suggest that distinctive substrate pools exist for Uba1 and Uba6 that reflect non-redundant biological roles for Uba6.

We have explored a general approach for the determination of absolute amounts and the relative stoichiometry of proteins in a mixture using fluorescence and mass spectrometry. We engineered a gene to express green fluorescent protein (GFP) with a synthetic fusion protein (GAB-GFP) in Escherichia coli to function as a spectroscopic standard for the quantification of an analogous stable isotope-labeled, non-fluorescent fusion protein (GAB*) and for the quantification and stoichiometric analysis of purified transducin, a heterotrimeric G-protein complex. Both GAB-GFP and GAB* contain concatenated sequences of specific proteotypic peptides that are derived from the ␣, ␤, and ␥ protein subunits of transducin and that are each flanked by spacer regions that maintain the native proteolytic properties for these peptide fragments. Spectroscopic quantification of GAB-GFP provided a molar scale for mass spectrometric ratios from tryptic peptides of GAB* and defined molar responses for mass spectrometric signal intensities from a purified transducin complex. The stoichiometry of transducin subunits ␣, ␤, and ␥ was measured to be 1:1.1:1.15 over a 5-fold range of labeled internal standard with a relative standard deviation of 9%. Fusing a unique genetically coded spectroscopic signal element with concatenated proteotypic peptides provides a powerful method to accurately quantify and determine the relative stoichiometry of multiple proteins present in complexes or mixtures that cannot be readily assessed using classical gravimetric, enzymatic, or antibody-based technologies. Molecular & Cellular Proteomics 7:442-447, 2008.We describe a general method to determine the stoichiometry and absolute quantification of proteins, a method that substantively extends previously developed techniques. Many proteins assemble to form protein complexes to fulfill unique functional roles. Protein assemblies provide a functional diversity that can contextually change with time and space during a cellular lifecycle. Many of these complexes have been dissected and catalogued in different organisms using the combination of tandem affinity purification followed by identification of individual members by mass spectrometry (1, 2). Comprehensive identification of individual members in protein assemblies is now performed routinely using mass spectrometry (3, 4). However, the determination of stoichiometry and quantification of individual proteins in complexes or mixtures usually requires the use of analytical ultracentrifugation.Several techniques for protein quantification using mass spectrometry depend on quantification of peptides generated during their proteolytic digestion. Chemical labeling of peptides after digestion (5) or metabolic labeling of growing cells in the presence of labeled substrate are useful for relative or comparative analyses of different sample groups (6). It has been proposed that the absolute quantification of proteins in complex protein mixtures can be accomplished by isotope dilution using synthetic labeled internal standa...

Duplication of transporter genes is apparent in the genome sequence of the hyperthermophilic bacterium Thermotoga maritima. The physiological impacts of these duplications are not well understood, so we used the bacterium's two putative maltose transporters to begin a study of the evolutionary relationship between a transporter's function and the control of expression of its genes. We show that the substrate binding proteins encoded by these operons, MalE1 and MalE2, have different substrate specificities and affinities and that they are expressed under different growth conditions. . Neither protein binds lactose. We examined the expression of these operons at both the transcriptional and translational levels and found that MalE1 is expressed in cells grown on lactose or guar gum and that MalE2 is highly expressed in starch-and trehalosegrown cells. Evidence is provided that malE1, malF1, and perhaps malG1 are cotranscribed and so constitute an operon. An open reading frame encoding a putative transcriptional regulatory protein adjacent to this operon (TM1200) is also up-regulated in response to growth on lactose. These evolutionarily related transporter operons have diverged both in function and expression to assume apparently different physiological roles.Annotation of the complete genome sequence of the hyperthermophilic, heterotrophic bacterium Thermotoga maritima indicates that 24% of the bacterium's open reading frames (ORFs) are most closely related to archaeal sequences (16). Subsequent studies have provided evidence that these genes were acquired by horizontal gene transfer (HGT). The closest known relative of T. maritima, Thermotoga species RQ2, acquired sugar ABC transporter and polysaccharide hydrolase genes independently of T. maritima, and this acquisition presumably provides selective nutritional advantages to this organism in its natural habitats (18). Intradomain gene acquisition or even gene duplication may confer novel selective advantages as well. This may be evident in the two putative maltose ABC transporters encoded in distant locations in the T. maritima genome (16). The amino acid sequences of the individual components of both transporters are very similar, suggesting a common evolutionary history (16). The transporter gene clusters (putative operons) may have arisen through operon duplication or horizontal acquisition of a second, orthologous operon by the ancestor of T. maritima. Evidence of a similar intradomain HGT of ABC transporter genes was found in two closely related archaea, Thermococcus litoralis and Pyrococcus furiosus (4). Regardless of their mechanism of duplication, the functions of these two T. maritima ABC transporters must confer distinct selective advantages since both have been retained.An organism will acquire a new catabolic trait after acquisition of new genes only if the genes are expressed and regulated in concert with other catabolism genes. Consequently, the evolution of the regions upstream of new genes and the binding of transcription factors to these regions...