Activity-Guided Proteomic Profiling of Proteasomes Uncovers a Variety of Active (and Inactive) Proteasome Species.

Proteasomes are multisubunit, multicatalytic protein complexes present in eukaryotic cells that degrade misfolded, damaged, or unstructured proteins. In this study, we used an activity-guided proteomic methodology based on a fluorogenic peptide substrate to characterize the composition of proteasome complexes in WT yeast and the changes these complexes undergo upon the deletion of Pre9 (Δα3) or of Sem1 (ΔSem1). A comparison of whole-cell proteomic analysis to activity-guided proteasome profiling indicates that the amounts of proteasomal proteins and proteasome interacting proteins in the assembled active proteasomes differ significantly from their total amounts in the cell as a whole. Using this activity-guided profiling approach, we characterized the changes in the abundance of subunits of various active proteasome species in different strains, quantified the relative abundance of active proteasomes across these strains, and charted the overall distribution of different proteasome species within each strain. The distributions obtained by our mass spectrometry-based quantification were markedly higher for some proteasome species than those obtained by activity-based quantification alone, suggesting that the activity of some of these species is impaired. The impaired activity appeared mostly among 20SBlm10 proteasome species which account for 20% of the active proteasomes in WT. To identify the factors behind this impaired activity, we mapped and quantified known proteasome-interacting proteins. Our results suggested that some of the reduced activity might be due to the association of the proteasome inhibitor Fub1. Additionally, we provide novel evidence for the presence of nonmature and therefore inactive proteasomal protease subunits ß2 and ß5 in the fully assembled proteasomes.

In-Depth Characterization of Apoptosis N-Terminome Reveals a Link Between Caspase-3 Cleavage and Posttranslational N-Terminal Acetylation.

The N termini of proteins contain information about their biochemical properties and functions. These N termini can be processed by proteases and can undergo other co- or posttranslational modifications. We have developed LATE (LysN Amino Terminal Enrichment), a method that uses selective chemical derivatization of α-amines to isolate the N-terminal peptides, in order to improve N-terminome identification in conjunction with other enrichment strategies. We applied LATE alongside another N-terminomic method to study caspase-3-mediated proteolysis both in vitro and during apoptosis in cells. This has enabled us to identify many unreported caspase-3 cleavages, some of which cannot be identified by other methods. Moreover, we have found direct evidence that neo-N-termini generated by caspase-3 cleavage can be further modified by Nt-acetylation. Some of these neo-Nt-acetylation events occur in the early phase of the apoptotic process and may have a role in translation inhibition. This has provided a comprehensive overview of the caspase-3 degradome and has uncovered previously unrecognized cross talk between posttranslational Nt-acetylation and caspase proteolytic pathways.

N-Terminomic Changes in Neurons During Excitotoxicity Reveal Proteolytic Events Associated With Synaptic Dysfunctions and Potential Targets for Neuroprotection.

Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIß (CaMKIIß). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIß, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.

A desert Chlorella sp. that thrives at extreme high-light intensities using a unique photoinhibition protection mechanism.

While light is the driving force of photosynthesis, excessive light can be harmful. Photoinhibition is one of the key processes that limit photosynthetic productivity. A well-defined mechanism that protects from photoinhibition has been described. Chlorella ohadii is a green micro-alga, isolated from biological desert soil crusts, which thrives under extreme high light (HL). Here, we show that this alga evolved unique protection mechanisms distinct from those of the green alga Chlamydomonas reinhardtii or plants. When grown under extreme HL, a drastic reduction in the size of light harvesting antennae occurs, resulting in the presence of core photosystem II, devoid of outer and inner antennas. This is accompanied by a massive accumulation of protective carotenoids and proteins that scavenge harmful radicals. At the same time, several elements central to photoinhibition protection in C. reinhardtii, such as psbS, light harvesting complex stress-related, photosystem II protein phosphorylation and state transitions are entirely absent or were barely detected. In addition, a carotenoid biosynthesis-related protein accumulates in the thylakoid membranes of HL cells and may function in sensing HL and protecting the cell from photoinhibition. Taken together, a unique photoinhibition protection mechanism evolved in C. ohadii, enabling the species to thrive under extreme-light intensities where other photosynthetic organisms fail to survive.

The protein phosphorylation landscape in photosystem I of the desert algae Chlorella sp.

The phosphorylation of photosystem II (PSII) and its antenna (LHCII) proteins has been studied, and its involvement in state transitions and PSII repair is known. Yet, little is known about the phosphorylation of photosystem I (PSI) and its antenna (LHCI) proteins. Here, we applied proteomics analysis to generate a map of the phosphorylation sites of the PSI-LHCI proteins in Chlorella ohadii cells that were grown under low or extreme high-light intensities (LL and HL). Furthermore, we analyzed the content of oxidized tryptophans and PSI-LHCI protein degradation products in these cells, to estimate the light-induced damage to PSI-LHCI. Our work revealed the phosphorylation of 17 of 22 PSI-LHCI subunits. The analyses detected the extensive phosphorylation of the LHCI subunits Lhca6 and Lhca7, which is modulated by growth light intensity. Other PSI-LHCI subunits were phosphorylated to a lesser extent, including PsaE, where molecular dynamic simulation proposed that a phosphoserine stabilizes ferredoxin binding. Additionally, we show that HL-grown cells accumulate less oxidative damage and degradation products of PSI-LHCI proteins, compared with LL-grown cells. The significant phosphorylation of Lhca6 and Lhca7 at the interface with other LHCI subunits suggests a physiological role during photosynthesis, possibly by altering light-harvesting characteristics and binding of other subunits.

Computational design of matrix metalloprotenaise-9 (MMP-9) resistant to auto-cleavage.

Matrix metalloproteinase-9 (MMP-9) is an endopeptidase that remodels the extracellular matrix. MMP-9 has been implicated in several diseases including neurodegeneration, arthritis, cardiovascular diseases, fibrosis and several types of cancer, resulting in a high demand for MMP-9 inhibitors for therapeutic purposes. For such drug design efforts, large amounts of MMP-9 are required. Yet, the catalytic domain of MMP-9 (MMP-9Cat) is an intrinsically unstable enzyme that tends to auto-cleave within minutes, making it difficult to use in drug design experiments and other biophysical studies. We set our goal to design MMP-9Cat variant that is active but stable to auto-cleavage. For this purpose, we first identified potential auto-cleavage sites on MMP-9Cat using mass spectroscopy and then eliminated the auto-cleavage site by predicting mutations that minimize auto-cleavage potential without reducing enzyme stability. Four computationally designed MMP-9Cat variants were experimentally constructed and evaluated for auto-cleavage and enzyme activity. Our best variant, Des2, with 2 mutations, was as active as the wild-type enzyme but did not exhibit auto-cleavage after 7 days of incubation at 37°C. This MMP-9Cat variant, with an identical with MMP-9Cat WT active site, is an ideal candidate for drug design experiments targeting MMP-9 and enzyme crystallization experiments. The developed strategy for MMP-9CAT stabilization could be applied to redesign other proteases to improve their stability for various biotechnological applications.

Granzyme B promotes cytotoxic lymphocyte transmigration via basement membrane remodeling.

Granzyme B (GzmB) is a protease with a well-characterized intracellular role in targeted destruction of compromised cells by cytotoxic lymphocytes. However, GzmB also cleaves extracellular matrix components, suggesting that it influences the interplay between cytotoxic lymphocytes and their environment. Here, we show that GzmB-null effector T cells and natural killer (NK) cells exhibited a cell-autonomous homing deficit in mouse models of inflammation and Ectromelia virus infection. Intravital imaging of effector T cells in inflamed cremaster muscle venules revealed that GzmB-null cells adhered normally to the vessel wall and could extend lamellipodia through it but did not cross it efficiently. In vitro migration assays showed that active GzmB was released from migrating cytotoxic lymphocytes and enabled chemokine-driven movement through basement membranes. Finally, proteomic analysis demonstrated that GzmB cleaved basement membrane constituents. Our results highlight an important role for GzmB in expediting cytotoxic lymphocyte diapedesis via basement membrane remodeling.

A novel recognition site for polyubiquitin and ubiquitin-like signals in an unexpected region of proteasomal subunit Rpn1.

The ubiquitin (Ub)-proteasome system is the primary mechanism for maintaining protein homeostasis in eukaryotes, yet the underlying signaling events and specificities of its components are poorly understood. Proteins destined for degradation are tagged with covalently linked polymeric Ub chains and subsequently delivered to the proteasome, often with the assistance of shuttle proteins that contain Ub-like domains. This degradation pathway is riddled with apparent redundancy-in the form of numerous polyubiquitin chains of various lengths and distinct architectures, multiple shuttle proteins, and at least three proteasomal receptors. Moreover, the largest proteasomal receptor, Rpn1, contains one known binding site for polyubiquitin and shuttle proteins, although several studies have recently proposed the existence of an additional uncharacterized site. Here, using a combination of NMR spectroscopy, photocrosslinking, mass spectrometry, and mutagenesis, we show that Rpn1 does indeed contain another recognition site that exhibits affinities and binding preferences for polyubiquitin and Ub-like signals comparable to those of the known binding site in Rpn1. Surprisingly, this novel site is situated in the N-terminal section of Rpn1, a region previously surmised to be devoid of functionality. We identified a stretch of adjacent helices as the location of this previously uncharacterized binding site, whose spatial proximity and similar properties to the known binding site in Rpn1 suggest the possibility of multivalent signal recognition across the solvent-exposed surface of Rpn1. These findings offer new mechanistic insights into signal recognition processes that are at the core of the Ub-proteasome system.

Phosphoproteomic characterization of the signaling network resulting from activation of the chemokine receptor CCR2.

Leukocyte recruitment is a universal feature of tissue inflammation and regulated by the interactions of chemokines with their G protein-coupled receptors. Activation of CC chemokine receptor 2 (CCR2) by its cognate chemokine ligands, including CC chemokine ligand 2 (CCL2), plays a central role in recruitment of monocytes in several inflammatory diseases. In this study, we used phosphoproteomics to conduct an unbiased characterization of the signaling network resulting from CCL2 activation of CCR2. Using data-independent acquisition MS analysis, we quantified both the proteome and phosphoproteome in FlpIn-HEK293T cells stably expressing CCR2 at six time points after activation with CCL2. Differential expression analysis identified 699 significantly regulated phosphorylation sites on 441 proteins. As expected, many of these proteins are known to participate in canonical signal transduction pathways and in the regulation of actin cytoskeleton dynamics, including numerous guanine nucleotide exchange factors and GTPase-activating proteins. Moreover, we identified regulated phosphorylation sites in numerous proteins that function in the nucleus, including several constituents of the nuclear pore complex. The results of this study provide an unprecedented level of detail of CCR2 signaling and identify potential targets for regulation of CCR2 function.

Integration of Two In-depth Quantitative Proteomics Approaches Determines the Kallikrein-related Peptidase 7 (KLK7) Degradome in Ovarian Cancer Cell Secretome.

Kallikrein-related peptidase 7 (KLK7) is a serine peptidase that is over expressed in ovarian cancer. In vitro functional analyses have suggested KLK7 to play a cancer progressive role, although monitoring of KLK7 expression has suggested a contradictory protective role for KLK7 in ovarian cancer patients. In order to help delineate its mechanism of action and thereby the functional roles, information on its substrate repertoire is crucial. Therefore, in this study a quantitative proteomics approach-PROtein TOpography and Migration Analysis Platform (PROTOMAP)-coupled with SILAC was used for in-depth analysis of putative KLK7 substrates from a representative ovarian cancer cell line, SKOV-3, secreted proteins. The Terminal Amine Isotopic Labeling of Substrates (TAILS) approach was used to determine the exact cleavage sites and to validate qPROTOMAP-identified putative substrates. By employing these two technically divergent approaches, exact cleavage sites on 16 novel putative substrates and two established substrates, matrix metalloprotease (MMP) 2 and insulin growth factor binding protein 3 (IGFBP3), were identified in the SKOV-3 secretome. Eight of these substrates were also identified on TAILS analysis of another ovarian cancer cell (OVMZ-6) secretome, with a further seven OVMZ-6 substrates common to the SKOV-3 qPROTOMAP profile. Identified substrates were significantly associated with the common processes of cell adhesion, extracellular matrix remodeling and cell migration according to the gene ontology (GO) biological process analysis. Biochemical validation supports a role for KLK7 in directly activating pro-MMP10, hydrolysis of IGFBP6 and cleavage of thrombospondin 1 with generation of a potentially bioactive N-terminal fragment. Overall, this study constitutes the most comprehensive analysis of the putative KLK7 degradome in any cancer to date, thereby opening new avenues for KLK7 research.

TRIM25 and DEAD-Box RNA Helicase DDX3X Cooperate to Regulate RIG-I-Mediated Antiviral Immunity.

The cytoplasmic retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) initiate interferon (IFN) production and antiviral gene expression in response to RNA virus infection. Consequently, RLR signalling is tightly regulated by both host and viral factors. Tripartite motif protein 25 (TRIM25) is an E3 ligase that ubiquitinates multiple substrates within the RLR signalling cascade, playing both ubiquitination-dependent and -independent roles in RIG-I-mediated IFN induction. However, additional regulatory roles are emerging. Here, we show a novel interaction between TRIM25 and another protein in the RLR pathway that is essential for type I IFN induction, DEAD-box helicase 3X (DDX3X). In vitro assays and knockdown studies reveal that TRIM25 ubiquitinates DDX3X at lysine 55 (K55) and that TRIM25 and DDX3X cooperatively enhance IFNB1 induction following RIG-I activation, but the latter is independent of TRIM25's catalytic activity. Furthermore, we found that the influenza A virus non-structural protein 1 (NS1) disrupts the TRIM25:DDX3X interaction, abrogating both TRIM25-mediated ubiquitination of DDX3X and cooperative activation of the IFNB1 promoter. Thus, our results reveal a new interplay between two RLR-host proteins that cooperatively enhance IFN-ß production. We also uncover a new and further mechanism by which influenza A virus NS1 suppresses host antiviral defence.

Determination of the small RNA GcvB regulon in the Gram-negative bacterial pathogen Pasteurella multocida and identification of the GcvB seed binding region.

Pasteurella multocida is a Gram-negative bacterium responsible for many important animal diseases. While a number of P. multocida virulence factors have been identified, very little is known about how gene expression and protein production is regulated in this organism. Small RNA (sRNA) molecules are critical regulators that act by binding to specific mRNA targets, often in association with the RNA chaperone protein Hfq. In this study, transcriptomic analysis of the P. multocida strain VP161 revealed a putative sRNA with high identity to GcvB from Escherichia coli and Salmonella enterica serovar Typhimurium. High-throughput quantitative liquid proteomics was used to compare the proteomes of the P. multocida VP161 wild-type strain, a gcvB mutant, and a GcvB overexpression strain. These analyses identified 46 proteins that displayed significant differential production after inactivation of gcvB, 36 of which showed increased production. Of the 36 proteins that were repressed by GcvB, 27 were predicted to be involved in amino acid biosynthesis or transport. Bioinformatic analyses of putative P. multocida GcvB target mRNAs identified a strongly conserved 10 nucleotide consensus sequence, 5'-AACACAACAT-3', with the central eight nucleotides identical to the seed binding region present within GcvB mRNA targets in E. coli and S. Typhimurium. Using a defined set of seed region mutants, together with a two-plasmid reporter system that allowed for quantification of sRNA-mRNA interactions, this sequence was confirmed to be critical for the binding of the P. multocida GcvB to the target mRNA, gltA.

Outer membrane vesicles from Neisseria gonorrhoeae target PorB to mitochondria and induce apoptosis.

Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea by evading innate immunity. Colonizing the mucosa of the reproductive tract depends on the bacterial outer membrane porin, PorB, which is essential for ion and nutrient uptake. PorB is also targeted to host mitochondria and regulates apoptosis pathways to promote infections. How PorB traffics from the outer membrane of N. gonorrhoeae to mitochondria and whether it modulates innate immune cells, such as macrophages, remains unclear. Here, we show that N. gonorrhoeae secretes PorB via outer membrane vesicles (OMVs). Purified OMVs contained primarily outer membrane proteins including oligomeric PorB. The porin was targeted to mitochondria of macrophages after exposure to purified OMVs and wild type N. gonorrhoeae. This was associated with loss of mitochondrial membrane potential, release of cytochrome c, activation of apoptotic caspases and cell death in a time-dependent manner. Consistent with this, OMV-induced macrophage death was prevented with the pan-caspase inhibitor, Q-VD-PH. This shows that N. gonorrhoeae utilizes OMVs to target PorB to mitochondria and to induce apoptosis in macrophages, thus affecting innate immunity.

Proteotranscriptomic Measurements of E6-Associated Protein (E6AP) Targets in DU145 Prostate Cancer Cells.

Prostate cancer is a common cause of cancer-related death in men. E6AP (E6-Associated Protein), an E3 ubiquitin ligase and a transcription cofactor, is elevated in a subset of prostate cancer patients. Genetic manipulations of E6AP in prostate cancer cells expose a role of E6AP in promoting growth and survival of prostate cancer cells in vitro and in vivo However, the effect of E6AP on prostate cancer cells is broad and it cannot be explained fully by previously identified tumor suppressor targets of E6AP, promyelocytic leukemia protein and p27. To explore additional players that are regulated downstream of E6AP, we combined a transcriptomic and proteomic approach. We identified and quantified 16,130 transcripts and 7,209 proteins in castration resistant prostate cancer cell line, DU145. A total of 2,763 transcripts and 308 proteins were significantly altered on knockdown of E6AP. Pathway analyses supported the known phenotypic effects of E6AP knockdown in prostate cancer cells and in parallel exposed novel potential links of E6AP with cancer metabolism, DNA damage repair and immune response. Changes in expression of the top candidates were confirmed using real-time polymerase chain reaction. Of these, clusterin, a stress-induced chaperone protein, commonly deregulated in prostate cancer, was pursued further. Knockdown of E6AP resulted in increased clusterin transcript and protein levels in vitro and in vivo Concomitant knockdown of E6AP and clusterin supported the contribution of clusterin to the phenotype induced by E6AP. Overall, results from this study provide insight into the potential biological pathways controlled by E6AP in prostate cancer cells and identifies clusterin as a novel target of E6AP.

N-Terminomics TAILS Identifies Host Cell Substrates of Poliovirus and Coxsackievirus B3 3C Proteinases That Modulate Virus Infection.

Enteroviruses encode proteinases that are essential for processing of the translated viral polyprotein. In addition, viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. Although some host protein substrates of enterovirus proteinases have been identified, the full repertoire of targets remains unknown. We used a novel quantitative in vitro proteomics-based approach, termed terminal amine isotopic labeling of substrates (TAILS), to identify with high confidence 72 and 34 new host protein targets of poliovirus and coxsackievirus B3 (CVB3) 3C proteinases (3Cpros) in HeLa cell and cardiomyocyte HL-1 cell lysates, respectively. We validated a subset of candidate substrates that are targets of poliovirus 3Cproin vitro including three common protein targets, phosphoribosylformylglycinamidine synthetase (PFAS), hnRNP K, and hnRNP M, of both proteinases. 3Cpro-targeted substrates were also cleaved in virus-infected cells but not noncleavable mutant proteins designed from the TAILS-identified cleavage sites. Knockdown of TAILS-identified target proteins modulated infection both negatively and positively, suggesting that cleavage by 3Cpro promotes infection. Indeed, expression of a cleavage-resistant mutant form of the endoplasmic reticulum (ER)-Golgi vesicle-tethering protein p115 decreased viral replication and yield. As the first comprehensive study to identify and validate functional enterovirus 3Cpro substrates in vivo, we conclude that N-terminomics by TAILS is an effective strategy to identify host targets of viral proteinases in a nonbiased manner.IMPORTANCE Enteroviruses are positive-strand RNA viruses that encode proteases that cleave the viral polyprotein into the individual mature viral proteins. In addition, viral proteases target host proteins in order to modulate cellular pathways and block antiviral responses in order to facilitate virus infection. Although several host protein targets have been identified, the entire list of proteins that are targeted is not known. In this study, we used a novel unbiased proteomics approach to identify ∼100 novel host targets of the enterovirus 3C protease, thus providing further insights into the network of cellular pathways that are modulated to promote virus infection.

Oncogenic ZEB2 activation drives sensitivity toward KDM1A inhibition in T-cell acute lymphoblastic leukemia.

Elevated expression of the Zinc finger E-box binding homeobox transcription factor-2 (ZEB2) is correlated with poor prognosis and patient outcome in a variety of human cancer subtypes. Using a conditional gain-of-function mouse model, we recently demonstrated that ZEB2 is an oncogenic driver of immature T-cell acute lymphoblastic leukemia (T-ALL), a heterogenic subgroup of human leukemia characterized by a high incidence of remission failure or hematological relapse after conventional chemotherapy. Here, we identified the lysine-specific demethylase KDM1A as a novel interaction partner of ZEB2 and demonstrated that mouse and human T-ALLs with increased ZEB2 levels critically depend on KDM1A activity for survival. Therefore, targeting the ZEB2 protein complex through direct disruption of the ZEB2-KDM1A interaction or pharmacological inhibition of the KDM1A demethylase activity itself could serve as a novel therapeutic strategy for this aggressive subtype of human leukemia and possibly other ZEB2-driven malignancies.

Proteomic Identification of Interferon-Induced Proteins with Tetratricopeptide Repeats as Markers of M1 Macrophage Polarization.

Macrophages, which accumulate in tissues during inflammation, may be polarized toward pro-inflammatory (M1) or tissue reparative (M2) phenotypes. The balance between these phenotypes can have a substantial influence on the outcome of inflammatory diseases such as atherosclerosis. Improved biomarkers of M1 and M2 macrophages would be beneficial for research, diagnosis, and monitoring the effects of trial therapeutics in such diseases. To identify novel biomarkers, we have characterized the global proteomes of THP-1 macrophages polarized to M1 and M2 states in comparison with unpolarized (M0) macrophages. M1 polarization resulted in increased expression of numerous pro-inflammatory proteins including the products of 31 genes under the transcriptional control of interferon regulatory factor 1 (IRF-1). In contrast, M2 polarization identified proteins regulated by components of the transcription factor AP-1. Among the most highly upregulated proteins under M1 conditions were the three interferon-induced proteins with tetratricopeptide repeats (IFITs: IFIT1, IFIT2, and IFIT3), which function in antiviral defense. Moreover, IFIT1, IFIT2, and IFIT3 mRNA were strongly upregulated in M1 polarized human primary macrophages and IFIT1 was also expressed in a subset of macrophages in aortic sinus and brachiocephalic artery sections from atherosclerotic ApoE-/- mice. On the basis of these results, we propose that IFITs may serve as useful markers of atherosclerosis and potentially other inflammatory diseases.

Structure-function analyses of a pertussis-like toxin from pathogenic Escherichia coli reveal a distinct mechanism of inhibition of trimeric G-proteins.

Pertussis-like toxins are secreted by several bacterial pathogens during infection. They belong to the AB5 virulence factors, which bind to glycans on host cell membranes for internalization. Host cell recognition and internalization are mediated by toxin B subunits sharing a unique pentameric ring-like assembly. Although the role of pertussis toxin in whooping cough is well-established, pertussis-like toxins produced by other bacteria are less studied, and their mechanisms of action are unclear. Here, we report that some extra-intestinal Escherichia coli pathogens (i.e. those that reside in the gut but can spread to other bodily locations) encode a pertussis-like toxin that inhibits mammalian cell growth in vitro We found that this protein, EcPlt, is related to toxins produced by both nontyphoidal and typhoidal Salmonella serovars. Pertussis-like toxins are secreted as disulfide-bonded heterohexamers in which the catalytic ADP-ribosyltransferase subunit is activated when exposed to the reducing environment in mammalian cells. We found here that the reduced EcPlt exhibits large structural rearrangements associated with its activation. We noted that inhibitory residues tethered within the NAD+-binding site by an intramolecular disulfide in the oxidized state dissociate upon the reduction and enable loop restructuring to form the nucleotide-binding site. Surprisingly, although pertussis toxin targets a cysteine residue within the α subunit of inhibitory trimeric G-proteins, we observed that activated EcPlt toxin modifies a proximal lysine/asparagine residue instead. In conclusion, our results reveal the molecular mechanism underpinning activation of pertussis-like toxins, and we also identified differences in host target specificity.

Identification of Novel Acinetobacter baumannii Type VI Secretion System Antibacterial Effector and Immunity Pairs.

The type VI secretion system (T6SS) is a macromolecular machine that delivers protein effectors into host cells and/or competing bacteria. The effectors may be delivered as noncovalently bound cargo of T6SS needle proteins (VgrG/Hcp/PAAR) or as C-terminal extensions of these proteins. Many Acinetobacter baumannii strains produce a T6SS, but little is known about the specific effectors or how they are delivered. In this study, we show that A. baumannii AB307-0294 encodes three vgrG loci, each containing a vgrG gene, a T6SS toxic effector gene, and an antitoxin/immunity gene. Each of the T6SS toxic effectors could kill Escherichia coli when produced in trans unless the cognate immunity protein was coproduced. To determine the role of each VgrG in effector delivery, we performed interbacterial competitive killing assays using A. baumannii AB307-0294 vgrG mutants, together with Acinetobacter baylyi prey cells expressing pairs of immunity genes that protected against two toxic effectors but not a third. Using this approach, we showed that AB307-0294 produces only three T6SS toxic effectors capable of killing A. baylyi and that each VgrG protein is specific for the carriage of one effector. Finally, we analyzed a number of A. baumannii genomes and identified significant diversity in the range of encoded T6SS VgrG and effector proteins, with correlations between effector types and A. baumannii global clone lineages.

Exploring the oncoproteomic response of human prostate cancer to therapeutic radiation using data-independent acquisition (DIA) mass spectrometry.

INTRODUCTION: The development of radioresistance in prostate cancer (PCa) is an important clinical issue and is still largely uninformed by personalized molecular characteristics. The aim of this study was to establish a platform that describes the early oncoproteomic response of human prostate tissue to radiation therapy (RT) using a prospective human tissue cohort. METHODS: Fresh and fixed transperineal biopsies from eight men with clinically localized tumors were taken prior to and 14 days following a single fraction of high-dose-rate brachytherapy. Quantitative protein analysis was achieved using an optimized protein extraction pipeline and subsequent data-independent acquisition mass spectroscopy (DIA-MS). Ontology analyses were used to identify enriched functional pathways, with the candidates further interrogated in formalin-fixed paraffin-embedded tissue biopsies from five additional patients. RESULTS: We obtained a mean coverage of 5660 proteins from fresh tissue biopsies; with the principal post-radiation change observed being an increase in levels amongst a total of 49 proteins exhibiting abundance changes. Many of these changes in abundance varied between patients and, typically to prostate cancer tissue, exhibited a high level of heterogeneity. Ontological analysis revealed the enrichment of the protein activation cascades of three immunological pathways: humoral immune response, leukocyte mediated immunity and complement activation. These were predominantly associated with the extracellular space. We validated significant expression differences in between 20% and 61% of these candidates using the separate fixed-tissue cohort and established their feasibility as an experimental tissue resource by acquiring quantitative data for a mean of 5152 proteins per patient. DISCUSSION: In this prospective study, we have established a sensitive and reliable oncoproteomic pipeline for the analysis of both fresh and formalin-fixed human PCa tissue. We identified multiple pathways known to be radiation-responsive and have established a powerful database of candidates and pathways with no current association with RT. This information may be beneficial in the advancement of personalized therapies and potentially, predictive biomarkers.