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The Burkholderia cepacia complex (Bcc) is a group of bacteria including opportunistic human pathogens. Immunocompromised individuals and cystic fibrosis patients are especially vulnerable to serious infections by these bacteria, motivating the search for compounds with antimicrobial activity against the Bcc. Ubonodin is a lasso peptide with promising activity against Bcc species, working by inhibiting RNA polymerase in susceptible bacteria. We constructed a library of over 90 000 ubonodin variants with 2 amino acid substitutions and used a high-throughput screen and next-generation sequencing to examine the fitness of the entire library, generating the most comprehensive data set on lasso peptide activity so far. This screen revealed information regarding the structure-activity relationship of ubonodin over a large sequence space. Remarkably, the screen identified one variant with not only improved activity compared to wild-type ubonodin but also a submicromolar minimum inhibitory concentration (MIC) against a clinical isolate of the Bcc member Burkholderia cenocepacia . Ubonodin and several of the variants identified in this study had lower MICs against certain Bcc strains than those of many clinically approved antibiotics. Finally, the large library size enabled us to develop DeepLasso, a deep learning model that can predict the RNAP inhibitory activity of an ubonodin variant., Competing Interests: The authors declare the following competing financial interest(s): A.J.L. has previously applied for a patent covering aspects of the antimicrobial activity of ubonodin., (© 2023 The Authors. Published by American Chemical Society.)

Flavohemoglobins, which are widely distributed in prokaryotes and eukaryotes, play key roles in oxygen (O 2 ) transport and nitric oxide (·NO) defense. Hmp is the flavohemoglobin of Escherichia coli , and here we report that the translational fusion of Hmp to the N-terminus of heterologous proteins increases their expression in E. coli . The effect required the fusion of the proteins, and was independent of both the O 2 -binding and catalytic activity of Hmp. Increased expression was at the translational level, likely to be downstream of initiation, and we observed that as little as the first 100 amino acids of Hmp were sufficient to boost protein production. These data demonstrate the potential of Hmp as an N-terminal fusion tag to increase protein yield, and suggest that the utility of bacterial hemoglobins to biotechnology goes beyond their O 2 transport and ·NO detoxification capabilities.

Natural products remain a critical source of medicines and drug leads. One of the most rapidly growing superclasses of natural products is RiPPs: ribosomally synthesized and posttranslationally modified peptides. RiPPs have rich and diverse bioactivities. This review highlights examples of the molecular mechanisms of action that underly those bioactivities. Particular emphasis is placed on RiPP/target interactions for which there is structural information. This detailed mechanism of action work is critical toward the development of RiPPs as therapeutics and can also be used to prioritize hits in RiPP genome mining studies., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

The tree-like pattern of the mammary epithelium is formed during puberty through a process known as branching morphogenesis. Although mammary epithelial branching is stochastic and generates an epithelial tree with a random pattern of branches, the global orientation of the developing epithelium is predictably biased along the long axis of the gland. Here, we combine analysis of pubertal mouse mammary glands, a three-dimensional (3D)-printed engineered tissue model, and computational models of morphogenesis to investigate the origin and the dynamics of the global bias in epithelial orientation during pubertal mammary development. Confocal microscopy analysis revealed that a global bias emerges in the absence of pre-aligned networks of type I collagen in the fat pad and is maintained throughout pubertal development until the widespread formation of lateral branches. Using branching and annihilating random walk simulations, we found that the angle of bifurcation of terminal end buds (TEBs) dictates both the dynamics and the extent of the global bias in epithelial orientation. Our experimental and computational data demonstrate that a local increase in stiffness from the accumulation of extracellular matrix, which constrains the angle of bifurcation of TEBs, is sufficient to pattern the global orientation of the developing mammary epithelium. These data reveal that local mechanical properties regulate the global pattern of mammary epithelial branching and may provide new insight into the global patterning of other branched epithelia., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Antibiotic resistance poses a serious threat to global health. To reinforce the anti-infective arsenal, many novel therapeutic strategies to fight bacterial infections are being explored. Among them, anti-virulence therapies, which target pathways important for virulence, have attracted much attention. Nitric oxide (NO) defense systems have been identified as critical for the pathogenesis of various bacteria, making them an appealing therapeutic target. In this study, we performed chemical screens to identify inhibitors of NO detoxification in Escherichia coli . We found that 2-mercaptobenzothiazole (2-MBT) can potently inhibit cellular detoxification of NO, achieving a level of inhibition that resembled the effect of genetically removing Hmp, the dominant detoxification enzyme under oxygenated conditions. Further analysis revealed that in the presence of NO, 2-MBT impaired the catalysis of Hmp and synthesis of Hmp and other proteins, whereas in its absence there were minimal perturbations to growth and protein synthesis. In addition, by studying the structure-activity relationship of 2-MBT, we found that both sulfur atoms in 2-MBT were vital for its inhibition of NO detoxification. Interestingly, when 2-mercaptothiazole (2-MT), which lacked the benzene ring, was used, differing biological activities were observed, although they too were NO dependent. Specifically, 2-MT could still prohibit NO detoxification, though it did not interfere with Hmp catalysis; rather, it was a stronger inhibitor of protein synthesis and it reduced the transcript levels of hmp , which was not observed with 2-MBT. Overall, these results provide a strong foundation for further exploration of 2-MBT and 2-MT for therapeutic applications., (Copyright © 2020 Chou, Vaikunthan, Schröder, Link, Kim and Brynildsen.)

Background: Post-database search is a key procedure in peptide identification with tandem mass spectrometry (MS/MS) strategies for refining peptide-spectrum matches (PSMs) generated by database search engines. Although many statistical and machine learning-based methods have been developed to improve the accuracy of peptide identification, the challenge remains on large-scale datasets and datasets with a distribution of unbalanced PSMs. A more efficient learning strategy is required for improving the accuracy of peptide identification on challenging datasets. While complex learning models have larger power of classification, they may cause overfitting problems and introduce computational complexity on large-scale datasets. Kernel methods map data from the sample space to high dimensional spaces where data relationships can be simplified for modeling., Results: In order to tackle the computational challenge of using the kernel-based learning model for practical peptide identification problems, we present an online learning algorithm, OLCS-Ranker, which iteratively feeds only one training sample into the learning model at each round, and, as a result, the memory requirement for computation is significantly reduced. Meanwhile, we propose a cost-sensitive learning model for OLCS-Ranker by using a larger loss of decoy PSMs than that of target PSMs in the loss function., Conclusions: The new model can reduce its false discovery rate on datasets with a distribution of unbalanced PSMs. Experimental studies show that OLCS-Ranker outperforms other methods in terms of accuracy and stability, especially on datasets with a distribution of unbalanced PSMs. Furthermore, OLCS-Ranker is 15-85 times faster than CRanker.

Over the course of roughly a decade, the lasso peptide field has been transformed. Whereas new compounds were discovered infrequently via activity-driven approaches, now, the vast majority of lasso peptide discovery is driven by genome-mining approaches. This paper starts with a historical overview of the first genome-mining approaches for lasso peptide discovery, and then covers new tools that have emerged. Several examples of novel lasso peptides that have been discovered via genome mining are presented as are examples of new enzymes found associated with lasso peptide gene clusters. Finally, this paper concludes with future directions and unsolved challenges in lasso peptide genome mining.

We report the identification of citrocin, a 19-amino acid-long antimicrobial lasso peptide from the bacteria Citrobacter pasteurii and Citrobacter braakii We refactored the citrocin gene cluster and heterologously expressed it in Escherichia coli We determined citrocin's NMR structure in water and found that is reminiscent of that of microcin J25 (MccJ25), an RNA polymerase-inhibiting lasso peptide that hijacks the TonB-dependent transporter FhuA to gain entry into cells. Citrocin has moderate antimicrobial activity against E. coli and Citrobacter strains. We then performed an in vitro RNA polymerase (RNAP) inhibition assay using citrocin and microcin J25 against E. coli RNAP. Citrocin has a higher minimal inhibition concentration than microcin J25 does against E. coli but surprisingly is ∼100-fold more potent as an RNAP inhibitor. This suggests that citrocin uptake by E. coli is limited. We found that unlike MccJ25, citrocin's activity against E. coli relied on neither of the two proton motive force-linked systems, Ton and Tol-Pal, for transport across the outer membrane. The structure of citrocin contains a patch of positive charge consisting of Lys-5 and Arg-17. We performed mutagenesis on these residues and found that the R17Y construct was matured into a lasso peptide but no longer had activity, showing the importance of this side chain for antimicrobial activity. In summary, we heterologously expressed and structurally and biochemically characterized an antimicrobial lasso peptide, citrocin. Despite being similar to MccJ25 in sequence, citrocin has an altered activity profile and does not use the same outer-membrane transporter to enter susceptible cells., (© 2019 Cheung-Lee et al.)

We report crystal structures of the antibacterial lasso peptides microcin J25 (MccJ25) and capistruin (Cap) bound to their natural enzymatic target, the bacterial RNA polymerase (RNAP). Both peptides bind within the RNAP secondary channel, through which NTP substrates enter the RNAP active site, and sterically block trigger-loop folding, which is essential for efficient catalysis by the RNAP. MccJ25 binds deep within the secondary channel in a manner expected to interfere with NTP substrate binding, explaining the partial competitive mechanism of inhibition with respect to NTPs found previously [Mukhopadhyay J, Sineva E, Knight J, Levy RM, Ebright RH (2004) Mol Cell 14:739-751]. The Cap binding determinant on RNAP overlaps, but is not identical to, that of MccJ25. Cap binds further from the RNAP active site and does not sterically interfere with NTP binding, and we show that Cap inhibition is partially noncompetitive with respect to NTPs. This work lays the groundwork for structure determination of other lasso peptides that target the bacterial RNAP and provides a structural foundation to guide lasso peptide antimicrobial engineering approaches., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Toxin-antitoxin (TA) systems interfere with essential cellular processes and are implicated in bacterial lifestyle adaptations such as persistence and the biofilm formation. Here, we present structural, biochemical, and functional data on an uncharacterized TA system, the COG5654-COG5642 pair. Bioinformatic analysis showed that this TA pair is found in 2,942 of the 16,286 distinct bacterial species in the RefSeq database. We solved a structure of the toxin bound to a fragment of the antitoxin to 1.50 Å. This structure suggested that the toxin is a mono-ADP-ribosyltransferase (mART). The toxin specifically modifies phosphoribosyl pyrophosphate synthetase (Prs), an essential enzyme in nucleotide biosynthesis conserved in all organisms. We propose renaming the toxin ParT for Prs ADP-ribosylating toxin and ParS for the cognate antitoxin. ParT is a unique example of an intracellular protein mART in bacteria and is the smallest known mART. This work demonstrates that TA systems can induce bacteriostasis through interference with nucleotide biosynthesis., Competing Interests: The authors declare no conflict of interest.

Background: Vaccine development for influenza A/H5N1 is an important public health priority, but H5N1 vaccines are less immunogenic than seasonal influenza vaccines. Adjuvant System 03 (AS03) markedly enhances immune responses to H5N1 vaccine antigens, but the underlying molecular mechanisms are incompletely understood., Objective and Methods: We compared the safety (primary endpoint), immunogenicity (secondary), gene expression (tertiary) and cytokine responses (exploratory) between AS03-adjuvanted and unadjuvanted inactivated split-virus H5N1 influenza vaccines. In a double-blinded clinical trial, we randomized twenty adults aged 18-49 to receive two doses of either AS03-adjuvanted (n = 10) or unadjuvanted (n = 10) H5N1 vaccine 28 days apart. We used a systems biology approach to characterize and correlate changes in serum cytokines, antibody titers, and gene expression levels in six immune cell types at 1, 3, 7, and 28 days after the first vaccination., Results: Both vaccines were well-tolerated. Nine of 10 subjects in the adjuvanted group and 0/10 in the unadjuvanted group exhibited seroprotection (hemagglutination inhibition antibody titer > 1:40) at day 56. Within 24 hours of AS03-adjuvanted vaccination, increased serum levels of IL-6 and IP-10 were noted. Interferon signaling and antigen processing and presentation-related gene responses were induced in dendritic cells, monocytes, and neutrophils. Upregulation of MHC class II antigen presentation-related genes was seen in neutrophils. Three days after AS03-adjuvanted vaccine, upregulation of genes involved in cell cycle and division was detected in NK cells and correlated with serum levels of IP-10. Early upregulation of interferon signaling-related genes was also found to predict seroprotection 56 days after first vaccination., Conclusions: Using this cell-based systems approach, novel mechanisms of action for AS03-adjuvanted pandemic influenza vaccination were observed., Trial Registration: ClinicalTrials.gov NCT01573312., Competing Interests: KME has received grant funding for group B streptococcal vaccine research from Novartis and has participated as a member on a Novartis Data Safety Monitoring Board on influenza vaccines. CBC has received institutional grant support for staphylococcal vaccine research from Pfizer and Novartis Vaccines. JBG, TJL, and HH are employed by The Emmes Corporation. The Emmes Corporation is an independent contract research organization NIH-NIAID-DMID contracted with for collecting and analyzing data for clinical trials and studies to evaluate the safety and efficacy of vaccines, devices, and therapeutics for infectious diseases. The Emmes Corporation does not have any patents, products in development or marketed products to declare. These affiliations do not alter our adherence to PLOS ONE policies on sharing data and materials; however, we have not released raw sequencing data to maintain accordance with subject confidentiality requirements as outlined in the study’s Informed Consent Document, as approved by the Vanderbilt University Medical Center Institutional Review Board. The other authors declare that they have no conflicts of interest.

Lasso peptides are a member of the superclass of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Like all RiPPs, lasso peptides are derived from a gene-encoded precursor protein. The biosynthesis of lasso peptides requires two enzymatic activities: proteolytic cleavage between the leader peptide and the core peptide in the precursor protein, accomplished by the B enzymes, and ATP-dependent isopeptide bond formation, accomplished by the C enzymes. In a subset of lasso peptide biosynthetic gene clusters from Gram-positive organisms, the B enzyme is split between two proteins. One such gene cluster is found in the organism Rhodococcus jostii , which produces the antimicrobial lasso peptide lariatin. The B enzyme in R. jostii is split between two open reading frames, larB1 and larB2 , both of which are required for lariatin biosynthesis. While the cysteine catalytic triad is found within the LarB2 protein, LarB1 is a PqqD homologue expected to bind to the lariatin precursor LarA based on its structural homology to other RiPP leader peptide binding domains. We show that LarB1 binds to the leader peptide of the lariatin precursor protein LarA with a sub-micromolar affinity. We used photocrosslinking with the noncanonical amino acid p -azidophenylalanine and mass spectrometry to map the interaction of LarA and LarB1. This analysis shows that the LarA leader peptide interacts with a conserved motif within LarB1 and, unexpectedly, the core peptide of LarA also binds to LarB1 in several positions. A Rosetta model built from distance restraints from the photocrosslinking experiments shows that the scissile bond between the leader peptide and core peptide in LarA is in a solvent-exposed loop.

Lasso peptide isopeptidase is an enzyme that specifically hydrolyzes the isopeptide bond of lasso peptides, rendering these peptides linear. To carry out a detailed structure-activity analysis of the lasso peptide isopeptidase AtxE2 from Asticcacaulis excentricus, we solved NMR structures of its substrates astexin-2 and astexin-3. Using in vitro enzyme assays, we show that the C-terminal tail portion of these peptides is dispensable with regards to isopeptidase activity. A collection of astexin-2 and astexin-3 variants with alanine substitutions at each position within the ring and the loop was constructed, and we showed that all of these peptides except for one were cleaved by the isopeptidase. Thus, much like the lasso peptide biosynthetic enzymes, lasso peptide isopeptidase has broad substrate specificity. Quantitative analysis of the cleavage reactions indicated that alanine substitutions in loop positions of these peptides led to reduced cleavage, suggesting that the loop is serving as a recognition element for the isopeptidase., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Ultimately, the genotype of a cell and its interaction with the environment determine the cell's biochemical state. While the cell's response to a single stimulus has been studied extensively, a conceptual framework to model the effect of multiple environmental stimuli applied concurrently is not as well developed. In this study, we developed the concepts of environmental interactions and epistasis to explain the responses of the S. cerevisiae proteome to simultaneous environmental stimuli. We hypothesize that, as an abstraction, environmental stimuli can be treated as analogous to genetic elements. This would allow modeling of the effects of multiple stimuli using the concepts and tools developed for studying gene interactions. Mirroring gene interactions, our results show that environmental interactions play a critical role in determining the state of the proteome. We show that individual and complex environmental stimuli behave similarly to genetic elements in regulating the cellular responses to stimuli, including the phenomena of dominance and suppression. Interestingly, we observed that the effect of a stimulus on a protein is dominant over other stimuli if the response to the stimulus involves the protein. Using publicly available transcriptomic data, we find that environmental interactions and epistasis regulate transcriptomic responses as well.

Extracellular signal-regulated kinase (ERK) coordinates cellular responses to a range of stimuli by phosphorylating its numerous substrates. One of these substrates, Capicua (Cic), is a transcriptional repressor that was first identified in Drosophila and has been implicated in a number of human diseases. Here we use a chemical biology approach to map the binding interface of ERK and Cic. The noncanonical amino acid p-azidophenylalanine (AzF) was introduced into the ERK-binding region of Drosophila Cic, and photocrosslinking and tandem mass spectrometry were used to pinpoint its binding site on ERK. We also identified the ERK-binding region of human Cic and showed that it binds to the same site on ERK despite lacking conservation with the Drosophila Cic binding region. Finally, we mapped the amino acids involved in human Cic binding to ERK using AzF-labeled ERK. These results reveal the molecular details of the ERK-Cic interaction and demonstrate that the photocrosslinking approach is complementary to existing methods for mapping kinase-substrate binding interfaces.

Systems biology is an approach to comprehensively study complex interactions within a biological system. Most published systems vaccinology studies have utilized whole blood or peripheral blood mononuclear cells (PBMC) to monitor the immune response after vaccination. Because human blood is comprised of multiple hematopoietic cell types, the potential for masking responses of under-represented cell populations is increased when analyzing whole blood or PBMC. To investigate the contribution of individual cell types to the immune response after vaccination, we established a rapid and efficient method to purify human T and B cells, natural killer (NK) cells, myeloid dendritic cells (mDC), monocytes, and neutrophils from fresh venous blood. Purified cells were fractionated and processed in a single day. RNA-Seq and quantitative shotgun proteomics were performed to determine expression profiles for each cell type prior to and after inactivated seasonal influenza vaccination. Our results show that transcriptomic and proteomic profiles generated from purified immune cells differ significantly from PBMC. Differential expression analysis for each immune cell type also shows unique transcriptomic and proteomic expression profiles as well as changing biological networks at early time points after vaccination. This cell type-specific information provides a more comprehensive approach to monitor vaccine responses.

Genome mining has unlocked a veritable treasure chest of natural compounds. However, each family of natural products requires a genome-mining approach tailored to its unique features to be successful. Lasso peptides are ribosomally synthesized and posttranslationally modified products with a unique three-dimensional structure. Advances in the understanding of these molecules have informed the design of strategies to identify new members of the class in sequenced genomes. This review presents the bioinformatic methods used to discover novel lasso peptides and describes how such analyses have afforded insights into the biosynthesis and evolution of this peptide class.

Background: The sequence database searching has been the dominant method for peptide identification, in which a large number of peptide spectra generated from LC/MS/MS experiments are searched using a search engine against theoretical fragmentation spectra derived from a protein sequences database or a spectral library. Selecting trustworthy peptide spectrum matches (PSMs) remains a challenge., Results: A novel scoring method named FC-Ranker is developed to assign a nonnegative weight to each target PSM based on the possibility of its being correct. Particularly, the scores of PSMs are updated by using a fuzzy SVM classification model and a fuzzy silhouette index iteratively. Trustworthy PSMs will be assigned high scores when the algorithm stops., Conclusions: Our experimental studies show that FC-Ranker outperforms other post-database search algorithms over a variety of datasets, and it can be extended to solve a general classification problem with uncertain labels.

A key step towards a chemical picture of enzyme catalysis was taken in 1913, when Leonor Michaelis and Maud Menten published their studies of sucrose hydrolysis by invertase. Based on a novel experimental design and a mathematical model, their work offered a quantitative view of biochemical kinetics well before the protein nature of enzymes was established and complexes with substrates could be detected. Michaelis-Menten kinetics provides a solid framework for enzyme kinetics in vitro, but what about kinetics in cells, where enzymes can be highly regulated and participate in a multitude of interactions? We discuss this question using the Extracellular Signal Regulated Kinase (ERK), which controls a myriad functions in cells, as a model of an important enzyme for which we have crystal structures, quantitative in vitro assays, and a vast list of binding partners. Despite great progress, we still cannot quantitatively predict how the rates of ERK-dependent reactions respond to genetic and pharmacological perturbations. Achieving this goal, which is important from both fundamental and practical standpoints, requires measuring the rates of enzyme reactions in their native environment and interpreting these measurements using simple but realistic mathematical models--the two elements which served as the cornerstones for Michaelis' and Menten's seminal 1913 paper., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

CD8+ T cells (TCD8) confer protective immunity against many infectious diseases, suggesting that microbial TCD8 determinants are promising vaccine targets. Nevertheless, current T cell antigen identification approaches do not discern which epitopes drive protective immunity during active infection - information that is critical for the rational design of TCD8-targeted vaccines. We employed a proteomics-based approach for large-scale discovery of naturally processed determinants derived from a complex pathogen, vaccinia virus (VACV), that are presented by the most frequent representatives of four major HLA class I supertypes. Immunologic characterization revealed that many previously unidentified VACV determinants were recognized by smallpox-vaccinated human peripheral blood cells in a variegated manner. Many such determinants were recognized by HLA class I-transgenic mouse immune TCD8 too and elicited protective TCD8 immunity against lethal intranasal VACV infection. Notably, efficient processing and stable presentation of immune determinants as well as the availability of naive TCD8 precursors were sufficient to drive a multifunctional, protective TCD8 response. Our approach uses fundamental insights into T cell epitope processing and presentation to define targets of protective TCD8 immunity within human pathogens that have complex proteomes, suggesting that this approach has general applicability in vaccine sciences.

Using affinity purifications coupled with mass spectrometry and yeast two-hybrid assays, we show the Saccharomyces cerevisiae translation initiation factor complex eukaryotic translation initiation factor 2B (eIF2B) and the very-long-chain fatty acid (VLCFA) synthesis keto-reductase enzyme YBR159W physically interact. The data show that the interaction is specifically between YBR159W and eIF2B and not between other members of the translation initiation or VLCFA pathways. A ybr159wΔ null strain has a slow-growth phenotype and a reduced translation rate but a normal GCN4 response to amino acid starvation. Although YBR159W localizes to the endoplasmic reticulum membrane, subcellular fractionation experiments show that a fraction of eIF2B cofractionates with lipid membranes in a YBR159W-independent manner. We show that a ybr159wΔ yeast strain and other strains with null mutations in the VLCFA pathway cause eIF2B to appear as numerous foci throughout the cytoplasm.

Lasso peptides are a class of ribosomally synthesized posttranslationally modified natural products found in bacteria. Currently known lasso peptides have a diverse set of pharmacologically relevant activities, including inhibition of bacterial growth, receptor antagonism, and enzyme inhibition. The biosynthesis of lasso peptides is specified by a cluster of three genes encoding a precursor protein and two enzymes. Here we develop a unique genome-mining algorithm to identify lasso peptide gene clusters in prokaryotes. Our approach involves pattern matching to a small number of conserved amino acids in precursor proteins, and thus allows for a more global survey of lasso peptide gene clusters than does homology-based genome mining. Of more than 3,000 currently sequenced prokaryotic genomes, we found 76 organisms that are putative lasso peptide producers. These organisms span nine bacterial phyla and an archaeal phylum. To provide validation of the genome-mining method, we focused on a single lasso peptide predicted to be produced by the freshwater bacterium Asticcacaulis excentricus. Heterologous expression of an engineered, minimal gene cluster in Escherichia coli led to the production of a unique lasso peptide, astexin-1. At 23 aa, astexin-1 is the largest lasso peptide isolated to date. It is also highly polar, in contrast to many lasso peptides that are primarily hydrophobic. Astexin-1 has modest antimicrobial activity against its phylogenetic relative Caulobacter crescentus. The solution structure of astexin-1 was determined revealing a unique topology that is stabilized by hydrogen bonding between segments of the peptide.

The channel pore-forming α subunit Kv4.2 is a major constituent of A-type (I(A)) potassium currents and a key regulator of neuronal membrane excitability. Multiple mechanisms regulate the properties, subcellular targeting, and cell-surface expression of Kv4.2-encoded channels. In the present study, shotgun proteomic analyses of immunoprecipitated mouse brain Kv4.2 channel complexes unexpectedly identified the voltage-gated Na⁺ channel accessory subunit Navβ1. Voltage-clamp and current-clamp recordings revealed that knockdown of Navβ1 decreases I(A) densities in isolated cortical neurons and that action potential waveforms are prolonged and repetitive firing is increased in Scn1b-null cortical pyramidal neurons lacking Navβ1. Biochemical and voltage-clamp experiments further demonstrated that Navβ1 interacts with and increases the stability of the heterologously expressed Kv4.2 protein, resulting in greater total and cell-surface Kv4.2 protein expression and in larger Kv4.2-encoded current densities. Together, the results presented here identify Navβ1 as a component of native neuronal Kv4.2-encoded I(A) channel complexes and a novel regulator of I(A) channel densities and neuronal excitability.

Cellulose degradation is essential for the future production of many advanced biofuels. Cellulases from the filamentous fungus Trichoderma reesei are among the most efficient enzymes for the hydrolysis of cellulosic materials. One of the cellulases from T. reesei, cellobiohydrolase II (CBH2), was studied because of its industrial relevance and proven enzymatic activity. Using both crude and rigorous membrane fractionation methods we show that full length T. reesei CBH2 is exclusively localized to the outer membrane when expressed recombinantly in Escherichia coli. Even fusing signal sequence-free maltose-binding protein to the N-terminus of CBH2, which has been shown to increase solubility of other proteins, did not prevent the outer membrane localization of CBH2. These results highlight the difficulties in producing fungal cellulases in bacterial hosts and provide a stepping stone for future cellulase engineering efforts.

Botulinum neurotoxin (BoNT) belongs to a large class of toxic proteins that act by enzymatically modifying cytosolic substrates within eukaryotic cells. The process by which a catalytic moiety is transferred across a membrane to enter the cytosol is not understood for any such toxin. BoNT is known to form pH-dependent pores important for the translocation of the catalytic domain into the cytosol. As a first step toward understanding this process, we investigated the mechanism by which the translocation domain of BoNT associates with a model liposome membrane. We report conditions that allow pH-dependent proteoliposome formation and identify a sequence at the translocation domain C terminus that is protected from proteolytic degradation in the context of the proteoliposome. Fluorescence quenching experiments suggest that residues within this sequence move to a hydrophobic environment upon association with liposomes. EPR analyses of spin-labeled mutants reveal major conformational changes in a distinct region of the structure upon association and indicate the formation of an oligomeric membrane-associated intermediate. Together, these data support a model of how BoNT orients with membranes in response to low pH.

The mammalian cryptome consists of bioactive peptides generated by the proteolysis of precursor proteins. It is speculated that the cryptide repertoire increases the complexity of the proteome by an order of magnitude. Cryptides have been found to function in a wide range of processes including neuronal signaling, antigen presentation, and the inflammatory response. Due to their potential as therapeutic agents, there has been an increasing interest in studying cryptides. In this review, we discuss different approaches for discovering these hidden peptides and how proteomic tools can be utilized to aid in their identification and characterization.

Microcin J25 (MccJ25) is a 21 amino acid (aa) ribosomally synthesized antimicrobial peptide with an unusual structure in which the eight N-terminal residues form a covalently cyclized macrolactam ring through which the remaining 13 aa tail is fed. An open question is the extent of sequence space that can occupy such an extraordinary, highly constrained peptide fold. To begin answering this question, here we have undertaken a computational redesign of the MccJ25 peptide using a two-stage sequence selection procedure based on both energy minimization and fold specificity. Eight of the most highly ranked sequences from the design algorithm, each of which contained two or three amino acid substitutions, were expressed in Escherichia coli and tested for production and antimicrobial activity. Six of the eight variants were successfully produced by E.coli at production levels comparable with that of the wild-type peptide. Of these six variants, three retain detectable antimicrobial activity, although this activity is reduced relative to wild-type MccJ25. The results here build upon previous findings that even rigid, constrained structures like the lasso architecture are amenable to redesign. Furthermore, this work provides evidence that a large amount of amino acid variation is tolerated by the lasso peptide fold.

Fundamental knowledge about how G protein-coupled receptors and their ligands interact is important for understanding receptor-ligand binding and the development of new drug discovery strategies. We have used cross-linking and tandem mass spectrometry analyses to investigate the interaction of the N terminus of the Saccharomyces cerevisiae tridecapeptide pheromone, α-factor (WHWLQLKPGQPMY), and Ste2p, its cognate G protein-coupled receptor. The Trp(1) residue of α-factor was replaced by 3,4-dihydroxyphenylalanine (DOPA) for periodate-mediated chemical cross-linking, and biotin was conjugated to Lys(7) for detection purposes to create the peptide [DOPA(1),Lys(7)(BioACA),Nle(12)]α-factor, called Bio-DOPA(1)-α-factor. This ligand analog was a potent agonist and bound to Ste2p with ∼65 nanomolar affinity. Immunoblot analysis of purified Ste2p samples that were treated with Bio-DOPA(1)-α-factor showed that the peptide analog cross-linked efficiently to Ste2p. The cross-linking was inhibited by the presence of either native α-factor or an α-factor antagonist. MALDI-TOF and immunoblot analyses revealed that Bio-DOPA(1)-α-factor cross-linked to a fragment of Ste2p encompassing residues Ser(251)-Met(294). Fragmentation of the cross-linked fragment and Ste2p using tandem mass spectrometry pinpointed the cross-link point of the DOPA(1) of the α-factor analog to the Ste2p Lys(269) side chain near the extracellular surface of the TM6-TM7 bundle. This conclusion was confirmed by a greatly diminished cross-linking of Bio-DOPA(1)-α-factor into a Ste2p(K269A) mutant. Based on these and previously obtained binding contact data, a mechanism of α-factor binding to Ste2p is proposed. The model for bound α-factor shows how ligand binding leads to conformational changes resulting in receptor activation of the signal transduction pathway.

The development of a robust and portable biosensor for the detection of pathogenic bacteria could impact areas ranging from water-quality monitoring to testing of pharmaceutical products for bacterial contamination. Of particular interest are detectors that combine the natural specificity of biological recognition with sensitive, label-free sensors providing electronic readout. Evolution has tailored antimicrobial peptides to exhibit broad-spectrum activity against pathogenic bacteria, while retaining a high degree of robustness. Here, we report selective and sensitive detection of infectious agents via electronic detection based on antimicrobial peptide-functionalized microcapacitive electrode arrays. The semiselective antimicrobial peptide magainin I--which occurs naturally on the skin of African clawed frogs--was immobilized on gold microelectrodes via a C-terminal cysteine residue. Significantly, exposing the sensor to various concentrations of pathogenic Escherichia coli revealed detection limits of approximately 1 bacterium/μL, a clinically useful detection range. The peptide-microcapacitive hybrid device was further able to demonstrate both Gram-selective detection as well as interbacterial strain differentiation, while maintaining recognition capabilities toward pathogenic strains of E. coli and Salmonella. Finally, we report a simulated "water-sampling" chip, consisting of a microfluidic flow cell integrated onto the hybrid sensor, which demonstrates real-time on-chip monitoring of the interaction of E. coli cells with the antimicrobial peptides. The combination of robust, evolutionarily tailored peptides with electronic read-out monitoring electrodes may open exciting avenues in both fundamental studies of the interactions of bacteria with antimicrobial peptides, as well as the practical use of these devices as portable pathogen detectors.

Unlabelled: Tumor microenvironment constitutes a reservoir for proteins released from tumor cells and the host, which can contribute significantly to tumor growth and invasion. This study aims to apply a method of combining in vivo microdialysis and proteomics to identify proteins in mammary tumor interstitial fluids, a major component of tumor microenvironment. In vivo microdialysis was performed in polyomavirus middle T antigen (PyVmT) transgenic mouse mammary tumors and age-matched control wild-type mammary glands. Over four hundred proteins were identified from the microdialysis perfusates, using the Multidimensional Protein Identification Technology. Osteopontin (OPN) is one of the proteins overexpressed in breast tumor perfusates, as confirmed with immunoassays. OPN was also found to be present in tumor-associated stroma in both PyVmT and human breast tumors, using immunohistochemistry. Specifically, fibroblasts were further shown to express OPN at both mRNA and protein levels. In vitro assays showed that OPN can stimulate PyVmT breast carcinoma cell proliferation and migration. Finally, the expression of OPN was significantly higher in the peripheral blood of mice bearing breast tumors, compared to wild-type mice. Overall, microdialysis combined with proteomics is a unique technique for identifying proteins in a tumor microenvironment in vivo. Mammary fibroblasts can secrete OPN, and its overexpression in mammary tumor microenvironment may contribute significantly to mammary tumor progression., Electronic Supplementary Material: The online version of this article (doi:10.1007/s12307-010-0046-3) contains supplementary material, which is available to authorized users.

Myotonic dystrophy types 1 and 2 (DM1 and DM2) are forms of muscular dystrophy that share similar clinical and molecular manifestations, such as myotonia, muscle weakness, cardiac anomalies, cataracts, and the presence of defined RNA-containing foci in muscle nuclei. DM2 is caused by an expansion of the tetranucleotide CCTG repeat within the first intron of ZNF9, although the mechanism by which the expanded nucleotide repeat causes the debilitating symptoms of DM2 is unclear. Conflicting studies have led to two models for the mechanisms leading to the problems associated with DM2. First, a gain-of-function disease model hypothesizes that the repeat expansions in the transcribed RNA do not directly affect ZNF9 function. Instead repeat-containing RNAs are thought to sequester proteins in the nucleus, causing misregulation of normal cellular processes. In the alternative model, the repeat expansions impair ZNF9 function and lead to a decrease in the level of translation. Here we examine the normal in vivo function of ZNF9. We report that ZNF9 associates with actively translating ribosomes and functions as an activator of cap-independent translation of the human ODC mRNA. This activity is mediated by direct binding of ZNF9 to the internal ribosome entry site sequence (IRES) within the 5'UTR of ODC mRNA. ZNF9 can activate IRES-mediated translation of ODC within primary human myoblasts, and this activity is reduced in myoblasts derived from a DM2 patient. These data identify ZNF9 as a regulator of cap-independent translation and indicate that ZNF9 activity may contribute mechanistically to the myotonic dystrophy type 2 phenotype.

The stress response of E. coli to the expression of two recombinant membrane proteins, the E. coli AAA+ protease FtsH and the human G-protein coupled receptor CB1, was examined using several members of a promoter-GFP library. Several genes from the heat-shock and envelope stress regulons (rpoH, clpP, lon, and ftsH) were strongly induced by expression of either membrane protein. Flow cytometry was used to monitor the real-time dynamics of the transcription of these reporter genes in response to membrane protein expression. Co-expression of CB1 and FtsH led to an additive response in these four reporter genes suggesting that the stresses may be occurring via different physiological mechanisms.

Somatodendritic A-type (I(A)) voltage-gated K(+) (K(V)) channels are key regulators of neuronal excitability, functioning to control action potential waveforms, repetitive firing and the responses to synaptic inputs. Rapidly activating and inactivating somatodendritic I(A) channels are encoded by K(V)4 alpha subunits and accumulating evidence suggests that these channels function as components of macromolecular protein complexes. Mass spectrometry (MS)-based proteomic approaches were developed and exploited here to identify potential components and regulators of native brain K(V)4.2-encoded I(A) channel complexes. Using anti-K(V)4.2 specific antibodies, K(V)4.2 channel complexes were immunoprecipitated from adult wild type mouse brain. Parallel control experiments were performed on brain samples isolated from (K(V)4.2(-/-)) mice harboring a targeted disruption of the KCND2 (K(V)4.2) locus. Three proteomic strategies were employed: an in-gel approach, coupled to one-dimensional liquid chromatography-tandem MS (1D-LC-MS/MS), and two in-solution approaches, followed by 1D- or 2D-LC-MS/MS. The targeted in-gel 1D-LC-MS/MS analyses demonstrated the presence of the K(V)4 alpha subunits (K(V)4.2, K(V)4.3 and K(V)4.1) and the K(V)4 accessory, KChIP (KChIP1-4) and DPP (DPP6 and 10), proteins in native brain K(V)4.2 channel complexes. The more comprehensive, in-solution approach, coupled to 2D-LC-MS/MS, also called Multidimensional Protein Identification Technology (MudPIT), revealed that additional regulatory proteins, including the K(V) channel accessory subunit K(V)beta1, are also components of native brain K(V)4.2 channel complexes. Additional biochemical and functional approaches will be required to elucidate the physiological roles of these newly identified K(V)4 interacting proteins.

Insig functions as a central regulator of cellular cholesterol homeostasis by controlling activity of HMG-CoA reductase (HMGR) in cholesterol synthesis. Insig both accelerates the degradation of HMGR and suppresses HMGR transcription through the SREBP-Scap pathway. The fission yeast Schizosaccharomyces pombe encodes homologs of Insig, HMGR, SREBP, and Scap, called ins1(+), hmg1(+), sre1(+), and scp1(+). Here, we characterize fission yeast Insig and demonstrate that Ins1 is dedicated to regulation of Hmg1, but not the Sre1-Scp1 pathway. Using a sterol-sensing domain mutant of Hmg1, we demonstrate that Ins1 binding to Hmg1 inhibits enzyme activity by promoting phosphorylation of the Hmg1 active site, which increases the K(M) for NADPH. Ins1-dependent phosphorylation of Hmg1 requires the MAP kinase Sty1/Spc1, and Hmg1 phosphorylation is physiologically regulated by nutrient stress. Thus, in fission yeast, Insig regulates sterol synthesis by a different mechanism than in mammalian cells, controlling HMGR phosphorylation in response to nutrient supply.

Yeast Mot1p, a member of the Snf2 ATPase family of proteins, is a transcriptional regulator that has the unusual ability to both repress and activate mRNA gene transcription. To identify interactions with other proteins that may assist Mot1p in its regulatory processes, Mot1p was purified from replicate yeast cell extracts, and Mot1p-associated proteins were identified by coupled multidimensional liquid chromatography and tandem mass spectrometry. Using this approach we generated a catalog of Mot1p-interacting proteins. Mot1p interacts with a range of transcriptional co-regulators as well as proteins involved in chromatin remodeling. We propose that interaction with such a wide range of proteins may be one mechanism through which Mot1p subserves its roles as a transcriptional activator and repressor.

Dyggve-Melchior-Clausen syndrome and Smith-McCort dysplasia are recessive spondyloepimetaphyseal dysplasias caused by loss-of-function mutations in dymeclin (Dym), a gene with previously unknown function. Here we report that Dym-deficient mice display defects in endochondral bone formation similar to that of Dyggve-Melchior-Clausen syndrome and Smith-McCort dysplasia, demonstrating functional conservation between the two species. Dym-mutant cells display multiple defects in vesicle traffic, as evidenced by enhanced dispersal of Golgi markers in interphase cells, delayed Golgi reassembly after brefeldin A treatment, delayed retrograde traffic of an endoplasmic reticulum-targeted Shiga toxin B subunit, and altered furin trafficking; and the Dym protein associates with multiple cellular proteins involved in vesicular traffic. These results establish dymeclin as a novel protein involved in Golgi organization and intracellular vesicle traffic and clarify the molecular basis for chondrodysplasia in mice and men.

G protein-coupled receptors (GPCRs) are notoriously difficult to express, particularly in microbial systems. Using GPCR fusions with the green fluorescent protein (GFP), we conducted studies to identify bacterial host effector genes that result in a general and significant enhancement in the amount of membrane-integrated human GPCRs that can be produced in Escherichia coli. We show that coexpression of the membrane-bound AAA+ protease FtsH greatly enhances the expression yield of four different class I GPCRs, irrespective of the presence of GFP. Using this new expression system, we produced 0.5 and 2 mg/L of detergent-solubilized and purified full-length central cannabinoid receptor (CB1) and bradykinin receptor 2 (BR2) in shake flask cultures, respectively, two proteins that had previously eluded expression in microbial systems.

Aims: Protein-protein interactions are critical for the normal membrane trafficking, localization, and function of voltage-gated ion channels. In human heart, the Shaker-related voltage-gated K(+) channel KCNA5 alpha-subunit forms the major basis of an atrial-specific, ultra-rapid delayed rectifier K(+) current, I(Kur). We sought to identify proteins that interact with KCNA5 in human atrium and investigate their role in the I(Kur) complex., Methods and Results: Using a glutathione-S-transferase (GST)-KCNA5 C-terminal fusion protein and mass spectrometry-based methods, the scaffolding protein four and a half LIM (for Lin-11, Isl-1, and Mec3) protein 1 (FHL1) was identified as a potential protein partner for KCNA5. Immunoprecipitation experiments confirmed a physical interaction of FHL1 with the K(+) channel complex in human atrium, as well as in Chinese hamster ovary (CHO) cells transfected with both KCNA5 and FHL1. In cotransfected cells, confocal microscopy demonstrated areas of colocalization after immunolabelling both proteins. To investigate the functional effects of this interaction, K(+) currents were recorded in CHO cells transfected with KCNA5 in the absence and presence of FHL1 coexpression. With coexpression of FHL1, K(+) current density was markedly increased, compared with cells expressing KCNA5 alone. This effect was associated with a shift in the voltage dependence of K(+) channel activation to more positive potentials, consistent with findings of I(Kur) in atrial myocytes. FHL1 also increased the extent and speed of K(+) current slow inactivation, with additional effects on the voltage dependence and recovery of this process., Conclusion: These results support a role of FHL1 as a key molecular component in the I(Kur) complex in human atrium, where it likely regulates functional expression of KCNA5.

The fluorescence of bacterial cells expressing a variant (GFPm) of the green fluorescent protein (GFP) was reduced to background levels by global replacement of the leucine residues of GFPm by 5,5,5-trifluoroleucine. Eleven rounds of random mutagenesis and screening via fluorescence-activated cell sorting yielded a GFP mutant containing 20 amino acid substitutions. The mutant protein in fluorinated form showed improved folding efficiency both in vivo and in vitro, and the median fluorescence of cells expressing the fluorinated protein was improved approximately 650-fold in comparison to that of cells expressing fluorinated GFPm. The success of this approach demonstrates the feasibility of engineering functional proteins containing many copies of abiological amino acid constituents.

The 5'-untranslated region of the ornithine decarboxylase (ODC) mRNA contains an internal ribosomal entry site (IRES). Mutational analysis of the ODC IRES has led to the identification of sequences necessary for cap-independent translation of the ODC mRNA. To discover novel IRES trans-acting factors (ITAFs), we performed a proteomics screen for proteins that regulate ODC translation using the wild-type ODC mRNA and a mutant version with an inactive IRES. We identified two RNA-binding proteins that associate with the wild-type ODC IRES but not the mutant IRES. One of these RNA-binding proteins, PCBP2, is an established activator of viral and cellular IRESs. The second protein, ZNF9 (myotonic dystrophy type 2 protein), has not been shown previously to bind IRES-like elements. Using a series of biochemical assays, we validated the interaction of these proteins with ODC mRNA. Interestingly ZNF9 and PCBP2 biochemically associated with each other and appeared to function as part of a larger holo-ITAF ribonucleoprotein complex. Our functional studies showed that PCBP2 and ZNF9 stimulate translation of the ODC IRES. Importantly these results may provide insight into the normal role of ZNF9 and why ZNF9 mutations cause myotonic dystrophy.

Epithelial-mesenchymal transition (EMT) is an important mechanism for phenotypic conversion in normal development and disease states such as tissue fibrosis and metastasis. While this conversion of epithelia is under tight transcriptional control, few of the key transcriptional proteins are known. Fibroblasts produced by EMT express a gene encoding fibroblast-specific protein 1 (FSP1), which is regulated by a proximal cis-acting promoter element called fibroblast transcription site-1 (FTS-1). In mass spectrometry, chromatin immunoprecipitation, and siRNA studies, we used FTS-1 as a unique probe for mediators of EMT and identified a complex of 2 proteins, CArG box-binding factor-A (CBF-A) and KRAB-associated protein 1 (KAP-1), that bind this site. Epithelial cells engineered to conditionally express recombinant CBF-A (rCBF-A) activate the transcription of FSP1 and undergo EMT. The FTS-1 response element also exists in the promoters modulating a broader EMT transcriptome, including Twist, and Snail, as well as E-cadherin, beta-catenin, ZO 1, vimentin, alpha1(I) collagen, and alpha-smooth muscle actin, and the induction of rCBF-A appropriately alters their expression as well. We believe formation of the CBF-A/KAP-1/FTS-1 complex is sufficient for the induction of FSP1 and a novel proximal activator of EMT.

Cytochrome P450 enzymes are heme-dependent monoxygenases that play a central role in human physiology. Despite the numerous physiological processes that P450 enzymes impact, the electron donors P450 oxidoreductase and cytochrome b5 are the only proteins known to interact with and modulate the activity of ER microsomal P450s. Here, we report that Dap1/PGRMC1 is required for ER P450 function in yeast and humans. We show that S. pombe Dap1 is a hemoprotein that binds and positively regulates Cyp51A1 and Cyp61A1, two P450s required for sterol biosynthesis. Similarly, loss of human PGRMC1 reduces activity of Cyp51A1, blocking cholesterol synthesis and increasing production of toxic sterol intermediates. PGRMC1 stably binds Cyp51A1 and human P450s from three additional families including Cyp3A4, which metabolizes pharmaceutical compounds. These findings demonstrate that PGRMC1 is required for P450 activity and suggest that interindividual variation in PGRMC1 function may impact multiple biochemical pathways and drug metabolism.

The anaphase-promoting complex/cyclosome (APC/C) is a conserved multisubunit ubiquitin ligase required for the degradation of key cell cycle regulators. The APC/C becomes active at the metaphase/anaphase transition and remains active during G(1) phase. One mechanism linked to activation of the APC/C is phosphorylation. Although many sites of mitotic phosphorylation have been identified in core components of the APC/C, the consequence of any individual phosphorylation event has not been elucidated in vivo. In this study, we show that Hcn1 is an essential core component of the fission yeast APC/C and is critical for maintaining complex integrity. Moreover, Hcn1 is a phosphoprotein in vivo. Phosphorylation of Hcn1 occurs at a single Cdk1 site in vitro and in vivo. Mutation of this site to alanine, but not aspartic acid, compromises APC/C function and leads to a specific defect in the completion of cell division.

Although generating large amounts of proteomic data using tandem mass spectrometry has become routine, there is currently no single set of comprehensive tools for the rigorous analysis of tandem mass spectrometry results given the large variety of possible experimental aims. Currently available applications are typically designed for displaying proteins and posttranslational modifications from the point of view of the mass spectrometrist and are not versatile enough to allow investigators to develop biological models of protein function, protein structure, or cell state. In addition, storage and dissemination of mass spectrometry-based proteomic data are problems facing the scientific community. To address these issues, we have developed a relational database model that efficiently stores and manages large amounts of tandem mass spectrometry results. We have developed an integrated suite of multifunctional analysis software for interpreting, comparing, and displaying these results. Our system, Bioinformatic Graphical Comparative Analysis Tools (BIGCAT), allows sophisticated analysis of tandem mass spectrometry results in a biologically intuitive format and provides a solution to many data storage and dissemination issues.

The incorporation of noncanonical amino acids into recombinant proteins in Escherichia coli can be facilitated by the introduction of new aminoacyl-tRNA synthetase activity into the expression host. We describe here a screening procedure for the identification of new aminoacyl-tRNA synthetase activity based on the cell surface display of noncanonical amino acids. Screening of a saturation mutagenesis library of the E. coli methionyl-tRNA synthetase (MetRS) led to the discovery of three MetRS mutants capable of incorporating the long-chain amino acid azidonorleucine into recombinant proteins with modest efficiency. The Leu-13 --> Gly (L13G) mutation is found in each of the three MetRS mutants, and the MetRS variant containing this single mutation is highly efficient in producing recombinant proteins that contain azidonorleucine.

In both normal and pathological states, cells respond rapidly to environmental cues by synthesizing new proteins. The selective identification of a newly synthesized proteome has been hindered by the basic fact that all proteins, new and old, share the same pool of amino acids and thus are chemically indistinguishable. We describe here a technology, based on the cotranslational introduction of azide groups into proteins and the chemoselective tagging of azide-labeled proteins with an alkyne affinity tag, to separate and identify, specifically, the newly synthesized proteins in mammalian cells. Incorporation of the azide-bearing amino acid azidohomoalanine is unbiased, not toxic, and does not increase protein degradation. As a first demonstration of the method, we report the selective purification and identification of 195 metabolically labeled proteins with multidimensional liquid chromatography in-line with tandem MS. Furthermore, in combination with leucine-based mass tagging, candidates were immediately validated as newly synthesized proteins. The identified proteins, synthesized in a 2-h window, possess a broad range of biochemical properties and span most functional gene ontology categories. This technology makes it possible to address the temporal and spatial characteristics of newly synthesized proteomes in any cell type.

Translation regulation is a critical means by which cells control growth, division, and apoptosis. To gain further insight into translation and related processes, we performed multifaceted mass spectrometry-based proteomic screens of yeast ribosomal complexes and discovered an association of 77 uncharacterized yeast proteins with ribosomes. Immunoblotting revealed an EDTA-dependent cosedimentation with ribosomes in sucrose gradients for 11 candidate translation-machinery-associated (TMA) proteins. Tandem affinity purification linked one candidate, LSM12, to the RNA processing proteins PBP1 and PBP4. A second candidate, TMA46, interacted with RBG1, a GTPase that interacts with ribosomes. By adapting translation assays to high-throughput screening methods, we showed that null yeast strains harboring deletions for several of the TMA genes had alterations in protein synthesis rates (TMA7 and TMA19), susceptibility to drugs that inhibit translation (TMA7), translation fidelity (TMA20), and polyribosome profiles (TMA7, TMA19, and TMA20). TMA20 has significant sequence homology with the oncogene MCT-1. Expression of human MCT-1 in the Deltatma20 yeast mutant complemented translation-related defects, strongly implying that MCT-1 functions in translation-related processes. Together these findings implicate the TMA proteins and, potentially, their human homologs, in translation related processes.

ARB1 is an essential yeast protein closely related to members of a subclass of the ATP-binding cassette (ABC) superfamily of proteins that are known to interact with ribosomes and function in protein synthesis or ribosome biogenesis. We show that depletion of ARB1 from Saccharomyces cerevisiae cells leads to a deficit in 18S rRNA and 40S subunits that can be attributed to slower cleavage at the A0, A1, and A2 processing sites in 35S pre-rRNA, delayed processing of 20S rRNA to mature 18S rRNA, and a possible defect in nuclear export of pre-40S subunits. Depletion of ARB1 also delays rRNA processing events in the 60S biogenesis pathway. We further demonstrate that ARB1 shuttles from nucleus to cytoplasm, cosediments with 40S, 60S, and 80S/90S ribosomal species, and is physically associated in vivo with TIF6, LSG1, and other proteins implicated previously in different aspects of 60S or 40S biogenesis. Mutations of conserved ARB1 residues expected to function in ATP hydrolysis were lethal. We propose that ARB1 functions as a mechanochemical ATPase to stimulate multiple steps in the 40S and 60S ribosomal biogenesis pathways.

Stimulated exocytosis of intracellular granules plays a critical role in conversion of inactive, circulating neutrophils to fully activated cells capable of chemotaxis, phagocytosis, and bacterial killing. The functional changes induced by exocytosis of each of the granule subsets, gelatinase (tertiary) granules, specific (secondary) granules, and azurophil (primary) granules, are poorly defined. To improve the understanding of the role of exocytosis of these granule subsets, a proteomic analysis of the azurophil, specific, and gelatinase granules from human neutrophils was performed. Two different methods for granule protein identification were applied. First, two-dimensional (2D) gel electrophoresis followed by MALDI-TOF MS analysis of peptides obtained by in-gel trypsin digestion of proteins was performed. Second, peptides from tryptic digests of granule membrane proteins were separated by two-dimensional microcapillary chromatography using strong cation exchange and reverse phase microcapillary high pressure liquid chromatography and analyzed with electrospray ionization tandem mass spectrometry (2D HLPC ESI-MS/MS). Our analysis identified 286 proteins on the three granule subsets, 87 of which were identified by MALDI MS and 247 were identified by 2D HPLC ESI-MS/MS. The increased sensitivity of 2D HPLC ESI-MS/MS, however, resulted in identification of over 500 proteins from subcellular organelles contaminating isolated granules. Defining the proteome of neutrophil granule subsets provides a basis for understanding the role of exocytosis in neutrophil biology. Additionally, the described methods may be applied to mobilizable compartments of other secretory cells.