Facile de Novo Sequencing of Tetrazine-Cyclized Peptides through UV-Induced Ring-Opening and Cleavage from the Solid Phase.

While most FDA-approved peptide drugs are cyclic, the robust cyclization chemistry of peptides and the deconvolution of cyclic peptide sequences by using tandem mass spectrometry render cyclic peptide drug discovery difficult. Here we present the successful design of cyclic peptides on solid phase that addresses both of these problems. We demonstrate that this peptide cyclization method using dichloro-s-tetrazine on solid phase allows successful cyclization of a panel of random peptide sequences with various charges and hydrophobicities. The cyclic peptides can be linearized and cleaved from the solid phase by simple UV light irradiation, and we demonstrate that accurate sequence information can be obtained for the UV-cleaved linearized peptides by using tandem mass spectrometry. The tetrazine linker used in the cyclic peptides can further be explored for inverse electron-demand Diels-Alder (IEDDA) reactions for screening or bioconjugation applications in the future.

Biomaterial Scaffolds as Pre-metastatic Niche Mimics Systemically Alter the Primary Tumor and Tumor Microenvironment.

Primary tumor (PT) immune cells and pre-metastatic niche (PMN) sites are critical to metastasis. Recently, synthetic biomaterial scaffolds used as PMN mimics are shown to capture both immune and metastatic tumor cells. Herein, studies are performed to investigate whether the scaffold-mediated redirection of immune and tumor cells would alter the primary tumor microenvironment (TME). Transcriptomic analysis of PT cells from scaffold-implanted and mock-surgery mice identifies differentially regulated pathways relevant to invasion and metastasis progression. Transcriptomic differences are hypothesized to result from scaffold-mediated modulations of immune cell trafficking and phenotype in the TME. Culturing tumor cells with conditioned media generated from PT immune cells of scaffold-implanted mice decrease invasion in vitro more than two-fold relative to mock surgery controls and reduce activity of invasion-promoting transcription factors. Secretomic characterization of the conditioned media delineates interactions between immune cells in the TME and tumor cells, showing an increase in the pan-metastasis inhibitor decorin and a concomitant decrease in invasion-promoting chemokine (C-C motif) ligand 2 (CCL2) in scaffold-implanted mice. Flow cytometric and transcriptomic profiling of PT immune cells identify phenotypically distinct tumor-associated macrophages (TAMs) in scaffold-implanted mice, which may contribute to an invasion-suppressive TME. Taken together, this study demonstrates biomaterial scaffolds systemically influence metastatic progression through manipulation of the TME.

Orthogonal ubiquitin transfer identifies ubiquitination substrates under differential control by the two ubiquitin activating enzymes.

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.

Dependence of the structure and mechanics of metaphase chromosomes on oxidized cysteines.

We have found that reagents that reduce oxidized cysteines lead to destabilization of metaphase chromosome folding, suggesting that chemically linked cysteine residues may play a structural role in mitotic chromosome organization, in accord with classical studies by Dounce et al. (J Theor Biol 42:275-285, 1973) and Sumner (J Cell Sci 70:177-188, 1984a). Human chromosomes isolated into buffer unfold when exposed to dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). In micromanipulation experiments which allow us to examine the mechanics of individual metaphase chromosomes, we have found that the gel-like elastic stiffness of native metaphase chromosomes is dramatically suppressed by DTT and TCEP, even before the chromosomes become appreciably unfolded. We also report protein labeling experiments on human metaphase chromosomes which allow us to tag oxidized and reduction-sensitive cysteine residues. PAGE analysis using fluorescent labels shows a small number of labeled bands. Mass spectrometry analysis of similarly labeled proteins provides a list of candidates for proteins with oxidized cysteines involved in chromosome organization, notably including components of condensin I, cohesin, the nucleosome-interacting proteins RCC1 and RCC2, as well as the RNA/DNA-binding protein NONO/p54NRB.

Extracellular matrix mediators of metastatic cell colonization characterized using scaffold mimics of the pre-metastatic niche.

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. STATEMENT OF SIGNIFICANCE: The pre-metastatic niche consists partially of ECM proteins that promote metastatic cell colonization to a target organ. We present a biomaterials-based approach to mimic this niche and identify ECM mediators of colonization. Using murine breast cancer models, we implanted microporous PCL scaffolds to recruit colonizing tumor cells in vivo. As a strategy to modulate colonization, we coated scaffolds with various ECM proteins, including decellularized lung and liver matrix from tumor-bearing mice. After characterizing the organ matrices using proteomics, myeloperoxidase was identified as an ECM protein contributing to colonization and validated using our scaffold. Our scaffold provides a platform to identify novel contributors to colonization and allows for the capture of colonizing tumor cells for a variety of downstream clinical applications.

Secretome identification of immune cell factors mediating metastatic cell homing.

Metastatic cell homing is a complex process mediated in part by diffusible factors secreted from immune cells found at a pre-metastatic niche. We report on connecting secretomics and TRanscriptional Activity CEll aRray (TRACER) data to identify functional paracrine interactions between immune cells and metastatic cells as novel mediators of homing. Metastatic breast cancer mouse models were used to generate a diseased splenocyte conditioned media (D-SCM) containing immune cell secreted factors. MDA-MB-231 metastatic cell activity including cell invasion, migration, transendothelial migration, and proliferation were increased in D-SCM relative to control media. Our D-SCM secretome analysis yielded 144 secreted factor candidates that contribute to increased metastatic cell activity. The functional mediators of homing were identified using MetaCore software to determine interactions between the immune cell secretome and the TRACER-identified active transcription factors within metastatic cells. Among the 5 candidate homing factors identified, haptoglobin was selected and validated in vitro and in vivo as a key mediator of homing. Our studies demonstrate a novel systems biology approach to identify functional signaling factors associated with a cellular phenotype, which provides an enabling tool that complements large-scale protein identification provided by proteomics.

α-Catenin phosphorylation promotes intercellular adhesion through a dual-kinase mechanism.

The cadherin-catenin adhesion complex is a key contributor to epithelial tissue stability and dynamic cell movements during development and tissue renewal. How this complex is regulated to accomplish these functions is not fully understood. We identified several phosphorylation sites in mammalian αE-catenin (also known as catenin α-1) and Drosophila α-Catenin within a flexible linker located between the middle (M)-region and the carboxy-terminal actin-binding domain. We show that this phospho-linker (P-linker) is the main phosphorylated region of α-catenin in cells and is sequentially modified at casein kinase 2 and 1 consensus sites. In Drosophila, the P-linker is required for normal α-catenin function during development and collective cell migration, although no obvious defects were found in cadherin-catenin complex assembly or adherens junction formation. In mammalian cells, non-phosphorylatable forms of α-catenin showed defects in intercellular adhesion using a mechanical dispersion assay. Epithelial sheets expressing phosphomimetic forms of α-catenin showed faster and more coordinated migrations after scratch wounding. These findings suggest that phosphorylation and dephosphorylation of the α-catenin P-linker are required for normal cadherin-catenin complex function in Drosophila and mammalian cells.

Direct binding of arsenic trioxide to AMPK and generation of inhibitory effects on acute myeloid leukemia precursors.

Arsenic trioxide (As2O3) exhibits potent antineoplastic effects and is used extensively in clinical oncology for the treatment of a subset of patients with acute myeloid leukemia (AML). Although As2O3 is known to regulate activation of several signaling cascades, the key events, accounting for its antileukemic properties, remain to be defined. We provide evidence that arsenic can directly bind to cysteine 299 in AMPKα and inhibit its activity. This inhibition of AMPK by arsenic is required in part for its cytotoxic effects on primitive leukemic progenitors from patients with AML, while concomitant treatment with an AMPK activator antagonizes in vivo the arsenic-induced antileukemic effects in a xenograft AML mouse model. A consequence of AMPK inhibition is activation of the mTOR pathway as a negative regulatory feedback loop. However, when AMPK expression is lost, arsenic-dependent activation of the kinase RSK downstream of MAPK activity compensates the generation of regulatory feedback signals through phosphorylation of downstream mTOR targets. Thus, therapeutic regimens with As2O3 will need to include inhibitors of both the mTOR and RSK pathways in combination to prevent engagement of negative feedback loops and maximize antineoplastic responses.

Multiple length peptide-pheromone variants produced by Streptococcus pyogenes directly bind Rgg proteins to confer transcriptional regulation.

Streptococcus pyogenes, a human-restricted pathogen, accounts for substantial mortality related to infections worldwide. Recent studies indicate that streptococci produce and respond to several secreted peptide signaling molecules (pheromones), including those known as short hydrophobic peptides (SHPs), to regulate gene expression by a quorum-sensing mechanism. Upon transport into the bacterial cell, pheromones bind to and modulate activity of receptor proteins belonging to the Rgg family of transcription factors. Previously, we reported biofilm regulation by the Rgg2/3 quorum-sensing circuit in S. pyogenes. The aim of this study was to identify the composition of mature pheromones from cell-free culture supernatants that facilitate biofilm formation. Bioluminescent reporters were employed to detect active pheromones in culture supernatants fractionated by reverse-phase chromatography, and mass spectrometry was used to characterize their properties. Surprisingly, multiple SHPs that varied by length were detected. Synthetic peptides of each variant were tested individually using bioluminescence reporters and biofilm growth assays, and although activities differed widely among the group, peptides comprising the C-terminal eight amino acids of the full-length native peptide were most active. Direct Rgg/SHP interactions were determined using a fluorescence polarization assay that utilized FITC-labeled peptide ligands. Peptide receptor affinities were seen to be as low as 500 nm and their binding affinities directly correlated with observed bioactivity. Revelation of naturally produced pheromones along with determination of their affinity for cognate receptors are important steps forward in designing compounds whose purpose is positioned for future therapeutics aimed at treating infections through the interference of bacterial communication.

Negatively charged metal oxide nanoparticles interact with the 20S proteasome and differentially modulate its biologic functional effects.

The multicatalytic ubiquitin-proteasome system (UPS) carries out proteolysis in a highly orchestrated way and regulates a large number of cellular processes. Deregulation of the UPS in many disorders has been documented. In some cases, such as carcinogenesis, elevated proteasome activity has been implicated in disease development, while the etiology of other diseases, such as neurodegeneration, includes decreased UPS activity. Therefore, agents that alter proteasome activity could suppress as well as enhance a multitude of diseases. Metal oxide nanoparticles, often developed as diagnostic tools, have not previously been tested as modulators of proteasome activity. Here, several types of metal oxide nanoparticles were found to adsorb to the proteasome and show variable preferential binding for particular proteasome subunits with several peptide binding "hotspots" possible. These interactions depend on the size, charge, and concentration of the nanoparticles and affect proteasome activity in a time-dependent manner. Should metal oxide nanoparticles increase proteasome activity in cells, as they do in vitro, unintended effects related to changes in proteasome function can be expected.

Sequence- and species-dependence of proteasomal processivity.

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 effects of chronic treatment with mood stabilizers on the rat hippocampal post-synaptic density proteome.

Bipolar disorder is a devastating illness that is marked by recurrent episodes of mania and depression. There is growing evidence that the disease is correlated with disruptions in synaptic plasticity cascades involved in cognition and mood regulation. Alleviating the symptoms of bipolar disorder involves chronic treatment with mood stabilizers like lithium or valproate. These two structurally dissimilar drugs are known to alter prominent signaling cascades in the hippocampus, but their effects on the post-synaptic density complex remain undefined. In this work, we utilized mass spectrometry for quantitative profiling of the rat hippocampal post-synaptic proteome to investigate the effects of chronic mood stabilizer treatment. Our data show that in response to chronic treatment of mood stabilizers there were not gross qualitative changes but rather subtle quantitative perturbations in post-synaptic density proteome linked to several key signaling pathways. Our data specifically support the changes in actin dynamics on valproate treatment. Using label-free quantification methods, we report that lithium and valproate significantly altered the abundance of 21 and 43 proteins, respectively. Seven proteins were affected similarly by both lithium and valproate: Ank3, glutamate receptor 3, dynein heavy chain 1, and four isoforms of the 14-3-3 family. Immunoblotting the same samples confirmed the changes in Ank3 and glutamate receptor 3 abundance. Our findings support the hypotheses that BPD is a synaptic disorder and that mood stabilizers modulate the protein signaling complex in the hippocampal post-synaptic density.

Evolution of mal ABC transporter operons in the Thermococcales and Thermotogales.

BACKGROUND: The mal genes that encode maltose transporters have undergone extensive lateral transfer among ancestors of the archaea Thermococcus litoralis and Pyrococcus furiosus. Bacterial hyperthermophiles of the order Thermotogales live among these archaea and so may have shared in these transfers. The genome sequence of Thermotoga maritima bears evidence of extensive acquisition of archaeal genes, so its ancestors clearly had the capacity to do so. We examined deep phylogenetic relationships among the mal genes of these hyperthermophiles and their close relatives to look for evidence of shared ancestry. RESULTS: We demonstrate that the two maltose ATP binding cassette (ABC) transporter operons now found in Tc. litoralis and P. furiosus (termed mal and mdx genes, respectively) are not closely related to one another. The Tc. litoralis and P. furiosus mal genes are most closely related to bacterial mal genes while their respective mdx genes are archaeal. The genes of the two mal operons in Tt. maritima are not related to genes in either of these archaeal operons. They are highly similar to one another and belong to a phylogenetic lineage that includes mal genes from the enteric bacteria. A unique domain of the enteric MalF membrane spanning proteins found also in these Thermotogales MalF homologs supports their relatively close relationship with these enteric proteins. Analyses of genome sequence data from other Thermotogales species, Fervidobacterium nodosum, Thermosipho melanesiensis, Thermotoga petrophila, Thermotoga lettingae, and Thermotoga neapolitana, revealed a third apparent mal operon, absent from the published genome sequence of Tt. maritima strain MSB8. This third operon, mal3, is more closely related to the Thermococcales' bacteria-derived mal genes than are mal1 and mal2. F. nodosum, Ts. melanesiensis, and Tt. lettingae have only one of the mal1-mal2 paralogs. The mal2 operon from an unknown species of Thermotoga appears to have been horizontally acquired by a Thermotoga species that had only mal1. CONCLUSION: These data demonstrate that the Tc. litoralis and P. furiosus mdx maltodextrin transporter operons arose in the Archaea while their mal maltose transporter operons arose in a bacterial lineage, but not the same lineage as the two maltose transporter operons found in the published Tt. maritima genome sequence. These Tt. maritima maltose transporters are phylogenetically and structurally similar to those found in enteric bacteria and the mal2 operon was horizontally transferred within the Thermotoga lineage. Other Thermotogales species have a third mal operon that is more closely related to the bacterial Thermococcales mal operons, but the data do not support a recent horizontal sharing of that operon between these groups.

Stoichiometry and absolute quantification of proteins with mass spectrometry using fluorescent and isotope-labeled concatenated peptide standards.

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 alpha, beta, and gamma 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 alpha, beta, and gamma 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.

Several archaeal homologs of putative oligopeptide-binding proteins encoded by Thermotoga maritima bind sugars.

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 beta-glucosides cellobiose and laminaribiose; TM0071 (XloE), xylobiose and xylotriose; TM0300 (GloE), large glucose oligosaccharides represented by xyloglucans; TM1223 (ManE), beta-1,4-mannobiose; and TM1226 (ManD), beta-1,4-mannobiose, beta-1,4-mannotriose, beta-1,4-mannotetraose, beta-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), alpha-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.

Substrate specificities and expression patterns reflect the evolutionary divergence of maltose ABC transporters in Thermotoga maritima.

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. MalE1 binds maltose (dissociation constant [KD], 24 +/- 1 microM), maltotriose (KD, 8 +/- 0.5 nM), and beta-(1-->4)-mannotetraose (KD, 38 +/- 1 microM). In contrast, MalE2 binds maltose (KD, 8.4 +/- 1 microM), maltotriose (KD, 11.5 +/- 1.5 microM), and trehalose (KD, 9.5 +/- 1.0 microM) confirming the findings of Wassenberg et al. (J. Mol. Biol. 295:279-288, 2000). 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 trehalose-grown 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.

Periplasmic maltose- and glucose-binding protein activities in cell-free extracts of Thermotoga maritima.

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 alpha-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 maltose-binding-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 maltose-binding proteins, MalE1 and MalE2, encoded in the genome. The maltose-binding 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.