Antibiotics are an important resource in modern medicine used to treat serious infections and enable a wide array of vital medical interventions including surgery and cancer chemotherapy. However, due to the increasing prevalence of antibiotic resistant pathogens, many clinically useful antibiotics are being rendered ineffective with too few new antibiotics in development to combat them. With highly diverse chemistry and bioactivity exquisitely shaped by evolution, natural products provide an unrivaled source of antibiotic compounds that is impossible to reproduce instinctively in the laboratory. The armeniaspirols are polyketide natural products with a unique spiro-[4.4]non-8-ene core that were isolated from Streptomyces armeniacus and were shown to be active against drug-resistant Gram-positive bacteria. Promisingly, in vitro resistant Staphylococcus aureus strains could not be readily obtained even after thirty serial passages under sub-lethal doses. Herein, we decipher the mechanism of action for this structurally unprecedented natural product antibiotic in the Gram-positive model organism Bacillus subtilis. Through chemical proteomics with an armeniaspirol-inspired activity-based probe, quantitative proteomics, biochemical assays, and microscopy, we show that armeniaspirol is a competitive inhibitor of the AAA+ proteases ClpXP and ClpYQ. Armeniaspirol represents the first known natural product inhibitor of ClpP, a highly coveted target due to its prominent role in bacterial virulence. Using overlapping proteomic fingerprints of armeniaspirol-treatment with ΔclpQ and ΔclpP deletions in B. subtilis, inhibition or deletion of these proteases appears to dysregulate key proteins involved in cell division, including FtsZ, DivIVA, and MreB. The dual ClpXP and ClpYQ inhibition is responsible for armeniaspirol’s potent antibiotic activity and this unique pharmacology makes it a promising candidate for antibiotic development. Several armeniaspirol-inspired analogs were generated as part of a medicinal chemistry study and evaluated for antibiotic activity towards a panel of clinically relevant Gram-positive pathogens. As a result, we identify three exciting armeniaspirol analogs with improved antibiotic activity. Lastly, the foundation for elucidating the ClpYQ degradome is developed. Our proteomic fingerprint of the B. subtilis ΔclpQ deletion strain generated some of the first insights into potential substrates of the ClpYQ protease. As a largely uncharacterized AAA+ protease implicated in the mechanism of action of armeniaspirol, we pursued a previously established acyl-intermediate covalent trapping strategy to characterize the ClpYQ-substrate complexes in B. subtilis cell lysate. Through unnatural amino acid incorporation using an evolved tRNA/aminoacyl-tRNA synthetase pair, the N-terminal active site serine of ClpQ is substituted with a photocleavable precursor that generates 2,3-diaminopropionic acid. While we were successful in synthesizing the photocleavable precursor, initial experiments to incorporate this unnatural amino acid in ClpQ expression proved unsuccessful, leading us to outline necessary control experiments for future endeavours. Ultimately, the covalently trapped substrates will be identified by LC-MS/MS, where we expect to identify key divisome and elongasome proteins in corroboration with the armeniaspirol mechanism of action study.

<p>There is a considerable body of evidence suggestive that dehydration can negatively impact voice production. However, our understanding of the underlying biology and physiological changes, particularly at the molecular level,  that contribute to this dysphonia are limited. Further, our ability to assess underlying changes in humans is restricted largely to post-mortem tissue or tissue resected during interventional vocal fold surgery, both of which are subject to bias in age and disease state. Here we have utilized a New Zealand white rabbit model of vocal fold dehydration to probe the <em>in vivo</em> molecular response to dehydration, focusing on differential gene and protein regulation. In the first study, a single 8-hour exposure to low humidity was used to induce airway surface dehydration. RNA Sequencing was used to obtain a global snapshot of differential transcriptional regulation. This informed a second study wherein 8-hour exposures to low humidity over 15 consecutive days were used and followed by LC-MS/MS proteomic analysis to interrogate potential functional changes. In the third study, systemic dehydration was induced with a 5-day water restriction protocol. A third rehydrated group was included that returned to <em>ad libitum</em> consumption for 3 days. LC-MS/MS proteomic analysis was used. We have found evidence for transcriptional and protein expression changes under both dehydration paradigms. Our findings serve to inform our molecular biological understanding of dehydration of the vocal folds with implications to prophylaxis against and clinical intervention thereof. </p>

<p>  </p> <p>Protein methylation is one of the most common protein posttranslational modifications (PTMs), within which protein α-N-terminal methylation is largely underexplored. Protein α-N-terminal methylation has been implicated in disease development, including cancer and neurodegenerative diseases, but the physiological and pathological roles of this PTM is not well understood. Protein α-N-terminal methylation modifies the free α-amino group on the protein N-termini and adds between one and three methyl groups by α-N-terminal methyltransferases. It has been shown that protein α-N-terminal methylation is conserved across prokaryotes and eukaryotes. The identification and characterization of the two α-N-terminal methyltransferases in humans, NTMT1 and NTMT2, and their homolog in yeast, Tae1, shows a high conserved substrate recognition and possible shared biological roles. α-N-terminal methyltransferases in humans and yeast recognize substrates with a canonical N-terminal motif, X1-P2-[K/R]3 (X=A, S, P or G after the initial M is cleaved). However, most of the proteins containing the canonical motif have not been studied and identified as substrates. In this study, we use a yeast as a model to explore the substrate members in the protein α-N-terminal methylome and understand the potential regulatory mechanisms. </p> <p>We characterized the yeast phenotypes associated with a <em>TAE1 </em>deletion strain, including increased resistance to heat stress, oxidative stress and paromomycin, and increased sensitivity to benomyl. We also extended the substrate repertoire by validating the presence of α-N-terminal methylation on six substrates by mass spectrometry. Furthermore, we investigate how α-N-terminal methylation could regulate Hsp31, a multifunctional heat shock protein that is associated with yeast heat response and oxidative response. Results suggest that methylation might regulate the localization of Hsp31, rather than directly regulating Hsp31 chaperone activity or methylglyoxalase activity. Alternatively, we developed another methodology to explore the α-N-terminal methylome without motif restriction by repurposing public mass spectrometry datasets for α-N-terminal methylation events in both yeast and humans. We found about 1-2 % of the total proteome are α-N-terminally methylated. Interestingly, the majority of the α-N-terminal methylation events were not on the canonical motif sequence. This indicates a more prevalent existence of α-N-terminal methylation.</p>

The overall goal of this series is to detail the paradigm shift that proteomics will bring to the practice of pediatric medicine and research. Proteomics is the global study of proteins in a biological system, tissue or bodily fluid. This first review will provide a brief overview of proteomics and describe its niche in the other "omics" of system biology. The underlying technology and methodology will be outlined as well as the obstacles that must be surmounted before pediatric proteomics is optimally useful for clinicians. The potential of proteomics in the area of personalized pediatric medicine will also be discussed since this is of particular clinical relevance. The second article in this series will focus on the application of proteomics to neonatology with particular emphasis on diseases where oxidative stress plays a key pathophysiological role.

Although the microcrustacean Daphnia is becoming an organism of choice for proteomic studies, protein expression across its life cycle have not been fully characterized. Proteomes of adult females, juveniles, asexually produced embryos, and the ephippia-resting stages containing sexually produced diapausing freezing- and desiccation-resistant embryos are analyzed. Overall, proteins with known molecular functions are more likely to be detected than proteins with no detectable orthology. Similarly, proteins with stronger gene model support in two independent genome assemblies can be detected, than those without such support. This suggests that the proteomics pipeline can be applied to verify hypothesized proteins, even given questionable reference gene models. In particular, upregulation of vitellogenins and downregulation of actins and myosins in embryos of both types, relative to juveniles and adults, and overrepresentation of cell-cycle related proteins in the developing embryos, relative to diapausing embryos and adults, are observed. Upregulation of small heat-shock proteins and peroxidases, as well as overrepresentation of stress-response proteins in the ephippium relative to the asexually produced non-diapausing embryos, is found. The ephippium also shows upregulation of three trehalose-synthesis proteins and downregulation of a trehalose hydrolase, consistent with the role of trehalose in protection against freezing and desiccation.

Tissues are heterogeneous ecosystems comprised of various cell types. For example, in tumor tissues, malignant cancer cells are surround by various non-malignant stromal cells. Proteins, especially N-linked glycoproteins, are key players in tumor microenvironment and respond to many extracellular stimuli for involving and regulating intercellular signaling. Understanding the human proteome and glycoproteome in heterogeneous tissues with spatial resolution are meaningful for exploring intercellular signaling networks and discovering protein biomarkers for various diseases, such as cancer. In this study, we aimed to develop new liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based analytical methods for spatially-resolved proteome and glycoproteome profiling in tissue samples, and apply them for profiling potential biomarkers for pancreatic cancer. We first systematically and synchronously optimized the LC-MS parameters to increase peptide sequencing efficiency in data dependent proteomics. Taking advantage of its hybrid instrument design with various mass analyzer and fragmentation strageties, the Orbitrap Fusion mass spectrometer was used for systematically comparing the popular high-high approach by using orbitrap for both MS1 and MS2 scans and high-low approach by using orbitrap for MS1 scan and ion trap for MS2 scans. High-high approach outperformed high-low approach in terms of better saturation of the scan cycle and higher MS2 identification rate. We then systematically optimized various MS parameters for high-high approach. We investigated the influence of isolation window and injection time on scan speed and MS2 identification rate. We then explored how to properly set dynamic exclusion time according to the chromatography peak width. Furthermore, we found that the orbitrap analyzer, rather than the analytical column, was easily saturated with higher peptide loading amount, thus limited the dynamic range of MS1-based quantification. Finally, by using the optimized LC-MS parameters, more than 9000 proteins and 110,000 unique peptides were identified by using 10 hours of effective LC gradient time. The study therefore illustrated the importance of synchronizing LC-MS precursor targeting and high-resolution fragment detection for high-efficient data dependent proteomics. Understanding the tumor heterogeneity through spatially resolved proteome profiling is meaningful for biomedical research. Laser capture microdissection (LCM) is a powerful technology for exploring local cell populations without losing spatial information. Here, we designed an immunohistochemistry (IHC)-based workflow for cell type-resolved proteome analysis of tissue samples. Firstly, targeted cell type was stained by IHC using antibody targeting cell-type specific marker to improve accuracy and efficiency of LCM. Secondly, to increase protein recovery from chemically crosslinked IHC tissues, we optimized a decrosslinking procedure to seamlessly combine with the integrated spintip-based sample preparation technology SISPROT. This newly developed approach, termed IHC-SISPROT, has comparable performance with traditional H&E staining-based proteomic analysis. High sensitivity and reproducibility of IHC-SISPROT was achieved by combining with data independent proteomic analysis. This IHC-SISPROT workflow was successfully applied for identifying 6660 and 6052 protein groups from cancer cells and cancer- associated fibroblasts (CAFs) by using only 5 mm 2 and 12 μm thickness of hepatocellular carcinoma tissue section. Bioinformatic analysis revealed the enrichment of cell type-specific ligands and receptors and potentially new communications between cancer cells and CAFs by these signaling proteins. Therefore, IHC-SISPROT is sensitive and accurate proteomic approach for spatial profiling of cell type-specific proteome from tissues. N-linked glycoproteins are promising candidates for diagnostic and prognostic biomarkers and therapeutic targets. They often locate at plasma membrane and extracellular space with distinct cell type distribution in tissue microenvironment. Due to access to only low microgram of proteins and low abundance of glycoproteins in tissue sections harvested by LCM, region- and cell type-resolved glycoproteome analysis of tissue sections remains challenging. Here we designed a fully integrated spintip-based glycoproteomic approach (FISGlyco) which achieved all the steps for glycoprotein enrichment, digestion, deglycosylation and desalting in a single spintip device. Sample loss is significantly reduced and the total processing time is reduced to 4 hours, while detection sensitivity and label-free quantification precision is greatly improved. 607 N-glycosylation sites were successfully identified and quantified from only 5 μg of mouse brain proteins. By seamlessly combining with LCM, the first region-resolved N-glycoproteome profiling of four mouse brain regions, including isocortex, hippocampus, thalamus, and hypothalamus, was achieved, with 1,875, 1,794, 1,801, and 1,417 N-glycosites identified, respectively. Our approach could be a generic approach for region and even cell type specific glycoproteome analysis of tissue sections. Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with five year survival rate of around 8%. No effective biomarkers and targeted therapy are one of the major reasons for this urgent clinical situation. To explore potential protein biomarkers and drug targets located at intercellular space of pancreatic tumor microenvironment, we established chemical proteomic approach for deep glycoproteome profiling of PDAC clinical tissue samples based on the above- mentioned new proteomic methods. Taking advantage of a long chain biotin- hydrazide probe with less space hindrance, the new method outperformed traditional hydrazide chemistry method in terms of sensitivity, time efficiency and glycoproteome coverage. The method was successfully applied to enrich and validate LIF and its receptors as potential biomarkers for PDAC. In addition, to explore the full map of pancreatic tumor microenvironment glycoproteome with diagnostic and therapeutic values, we collected 114 pancreatic tissues, including 30 PDAC tumor tissues, 30 adjacent non-tumor (NT) tissues, 32 chronic pancreatitis tissues and 22 normal pancreatic tissues, and systematically profiled their glycoprotein expression pattern by using the developed glycoproteomic strategy. The deepest glycoproteome of PDAC was achieved, which covered the majority of previously reported glycoprotein biomarkers and drug targets for PDAC. Importantly, we discovered many new glycoproteins with differential expression in PDAC and normal tissue types. Moreover, LCM-based cell-type proteome profiling was achieved for 13 PDAC tissue samples, which covered more than 8000 proteins for both pancreatic stromal cells and pancreatic cancer cells in each sample. We therefore provided a valuable resource for screening novel and cancer specific glycoprotein biomarkers for pancreatic cancer with spatial resolution

Le chloroplaste est un composant majeur de la cellule végétale. Cet organite est le fruit d’une endosymbiose, survenue entre une cellule eucaryote et une cyanobactérie. Ainsi, 95% des gènes codant pour les protéines plastidiales ont été transférés vers le génome nucléaire au cours de l’évolution. En conséquence, la plupart des protéines chloroplastiques sont aujourd’hui codées par le noyau, synthétisées dans le cytosol sous forme de précurseurs dotés d’une une extension N-terminale clivable (le "peptide de transit") et ensuite importées sans les chloroplastes via le système TOC/TIC (Translocons localisés au niveau des membranes externe et interne de l'enveloppe des chloroplastes). Jusqu'à récemment, toutes les protéines destinées aux compartiments chloroplastiques internes étaient censées posséder une séquence d’adressage N-terminale clivable et engager la machinerie d’import général TOC/TIC. Cependant, des études récentes reposant sur des approches protéomiques ont révélé l’existence de plusieurs protéines chloroplastiques dépourvues de la séquence additionnelle clivable. La première évidence de telles protéines dites non canoniques a été fournie par notre équipe, étudiant le protéome de l’enveloppe du chloroplaste d’Arabidopsis, qui a conduit à l’identification d’une protéine quinone oxidoréductase homologue nommée « ceQORH ». Bien que dépourvues de peptide de transit clivable, il s’est avéré que ces protéines sont capables de rejoindre les compartiments chloroplastiques internes. D’autre part, il a été également montré que l’import de ces protéines dans le chloroplaste n’est pas médiée par la machinerie de translocation générale TOC/TIC. De plus, il s’est avéré que ces protéines ont la particularité d’être multilocalisées dans les cellules de différents tissus de la feuille. Cependant, les mécanismes moléculaires qui contrôlent la localisation sub-cellulaire de telles protéines chloroplastiques non canoniques demeurent encore inconnus. Pour mieux caractériser fonctionnellement les composantes des systèmes d’import alternatifs de protéines chloroplastiques non canoniques, nous avons adopté une approche directe qui reposait sur des techniques biochimiques combinant le crosslink chimique, la purification par affinité et la spectrométrie de masse. Cette stratégie nous a permis d’identifier un partenaire, impliqué dans le contrôle de l’adressage de la protéine ceQORH dans le chloroplaste. Alternativement, nous avons réalisé une bio-analyse du protéome de l’enveloppe du chloroplaste et qui nous a permis de revisiter la composition du protéome de l’enveloppe du chloroplaste. Afin d’expliquer la localisation sub-cellulaire variable de la protéine ceQORH, les membres de l’équipe ont émis l’hypothèse d’une interaction probable de cette protéine avec un partenaire cytosolique. Dans la dernière partie de cette étude, nous avons validé l’interaction, in planta, entre ceQORH et son partenaire par une approche génétique qui portait sur l’analyse de l’impact de l’absence de ce dernier sur la régulation de la localisation sub-cellulaire de la protéine ceQORH. / Chloroplasts are a major component of plant cells. Their origin traces back to a cyanobacterial ancestor that was engulfed by an ancient eukaryotic cell and eventually integrated as an organelle during evolution. As a result, more than 95% of the ancestral cyanobacterial genes were transferred to the host cell nucleus. Proteins encoded by these relocated genes need to return to internal chloroplast compartments. This import is mainly achieved by the general TOC/TIC machinery located at the chloroplast surface. Until recently, all proteins destined to chloroplast were believed to possess an N-terminal and cleavable chloroplast targeting peptide, and to engage the TOC/TIC machinery. However, recent studies have revealed the existence of several non-canonical preproteins, lacking cleavable transit peptides. The first evidence for such ‘non-canonical’ chloroplast proteins was provided by our team studying the Arabidopsis chloroplast envelope proteome, leading to the identification of a quinone oxidoreductase homologue termed « ceQORH ». Furthermore, a few such proteins were demonstrated to use alternative targeting pathways, independent of the TOC/TIC machinery. To better characterize components of such alternative targeting machineries, a targeted study combining affinity purification and mass spectrometry aiming to identify alternative receptors at the chloroplast surface has been performed. This study allowed us to identify new “partner” involved in the control of chloroplast targeting of ceQORH protein. Alternatively, we also revisited the chloroplast envelope proteome composition and initiated a gene candidate approach. In addition, some non-canonical proteins are shared by plastids and other cell compartments. However, molecular mechanisms controlling subcellular localization of these non-canonical plastid proteins remain unknown. In order to explain the variable subcellular localization of ceQORH protein, our team hypothesized a probable interaction of ceQORH with a cytosolic partner. In the last part of this study, we validated the interaction between ceQORH and its partner in planta by a genetic approach analyzing the impact of the absence of the cytosolic partner on the regulation of the sub-cellular localization of ceQORH protein.

Indiana University-Purdue University Indianapolis (IUPUI) / RNA Polymerase II (Pol II) is the primary actor in the transcription of mRNA from genes. Pol II is a complex composed of twelve protein subunits. This study focused on the changes in the interactome of Rbp3 in S. cerevisiae when the Pol II subunit Rpb9 is removed. Rpb3 is one of the core subunits of Pol II, and any significant changes to the Rpb3 incteractome due to the loss of Rpb9 can be used to infer new information about Rpb9’s role in the Pol II complex. Rpb3 was pulled down using FLAG purification from both wild type and rpb9Δ S. cerevisiae cultures. Rpb3 and the proteins complexed with it were then analyzed using multi-dimensional protein identification technology (MudPIT), a form of liquid chromatography-mass spectrometry (LC-MS). This data was searched using the SEQUEST database search algorithm, and the results were further analyzed for likelihood of interaction using Significance Analysis of INTeractome (SAINT), as well as for post-translational phosphorylation. Deletion of rpb9 did not present any changes in Pol II phosphorylation however it did cause several changes in the interaction network. The rpb9Δ strain showed new interactions with Rtr1, Sen1, Vtc4, Pyc1, Tgl4, Sec61, Tfb2, Hfd1, Erv25, Rib4, Sla1, Ubp15, Bbc1, and Hxk1. The most prominent of these hits are Rtr1, an Rpb1 C-terminal domain phosphatase linked to transcription termination, and Sen1, an RNA/DNA nuclease that terminates transcription. In addition, this mutant showed no interaction with Mtd1, an interaction that is present in the wild type. In all cases, these hits should be considered fuel for future research, rather than conclusive evidence of novel interactions.

Indiana University-Purdue University Indianapolis (IUPUI) / In the forensic science community, there is a vast need for tools to help assist investigations when standard DNA profiling methods are uninformative. Methods such as Forensic DNA Phenotyping (FDP) and proteomics aims to help this problem and provide aid in investigations when other methods have been exhausted. FDP is useful by providing physical appearance information, while proteomics allows for the examination of difficult samples, such as hair, to infer human identity and ancestry. To create a “biological eye witness” or develop informative probability of identity match statistics through proteomically inferred genetic profiles, it is necessary to constantly strive to improve these methods. Currently, two developmentally validated FDP prediction assays, ‘HIrisPlex’ and ‘HIrisplex-S’, are used on the capillary electrophoresis to develop a phenotypic prediction for eye, hair, and skin color based on 41 variants. Although highly useful, these assays are limited in their ability when used on the CE due to a 25 variant per assay cap. To overcome these limitations and expand the capacities of FDP, we successfully designed and validated a massive parallel sequencing (MPS) assay for use on both the ThermoFisher Scientific Ion Torrent and Illumina MiSeq systems that incorporates all HIrisPlex-S variants into one sensitive assay. With the migration of this assay to an MPS platform, we were able to create a semi-automated pipeline to extract SNP-specific sequencing data that can then be easily uploaded to the freely accessible online phenotypic prediction tool (found at https://hirisplex.erasmusmc.nl) and a mixture deconvolution tool with built-in read count thresholds. Based on sequencing reads counts, this tool can be used to assist in the separation of difficult two-person mixture samples and outline the confidence in each genotype call. In addition to FDP, proteomic methods, specifically in hair protein analysis, opens doors and possibilities for forensic investigations when standard DNA profiling methods come up short. Here, we analyzed 233 genetically variant peptides (GVPs) within hair-associated proteins and genes for 66 individuals. We assessed the proteomic methods ability to accurately infer and detect genotypes at each of the 233 SNPs and generated statistics for the probability of identity (PID). Of these markers, 32 passed all quality control and population genetics criteria and displayed an average PID of 3.58 x 10-4. A population genetics assessment was also conducted to identify any SNP that could be used to infer ancestry and/or identity. Providing this information is valuable for the future use of this set of markers for human identification in forensic science settings.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19752号 / 工博第4207号 / 新制||工||1649(附属図書館) / 32788 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 濵地 格, 教授 梅田 眞郷, 教授 跡見 晴幸 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM