Robert L. J. Graham

Structural basis for high affinity binding of LEDGF PWWP to mononucleosomes

Lens epithelium-derived growth factor (LEDGF/p75) tethers lentiviral preintegration complexes (PICs) to chromatin and is essential for effective HIV-1 replication. LEDGF/p75 interactions with lentiviral integrases are well characterized, but the structural basis for how LEDGF/p75 engages chromatin is unknown. We demonstrate that cellular LEDGF/p75 is tightly bound to mononucleosomes (MNs). Our proteomic experiments indicate that this interaction is direct and not mediated by other cellular factors. We determined the solution structure of LEDGF PWWP and monitored binding to the histone H3 tail containing trimethylated Lys36 (H3K36me3) and DNA by NMR. Results reveal two distinct functional interfaces of LEDGF PWWP: a well-defined hydrophobic cavity, which selectively interacts with the H3K36me3 peptide and adjacent basic surface, which non-specifically binds DNA. LEDGF PWWP exhibits nanomolar binding affinity to purified native MNs, but displays markedly lower affinities for the isolated H3K36me3 peptide and DNA. Furthermore, we show that LEDGF PWWP preferentially and tightly binds to in vitro reconstituted MNs containing a tri-methyl-lysine analogue at position 36 of H3 and not to their unmodified counterparts. We conclude that cooperative binding of the hydrophobic cavity and basic surface to the cognate histone peptide and DNA wrapped in MNs is essential for high-affinity binding to chromatin.

Cell-specific proteomic analysis in Caenorhabditis elegans

Significance The emergence of mass spectrometry-based proteomics has revolutionized the study of proteins and their abundances, functions, interactions, and modifications. However, it is difficult to monitor dynamic changes in protein synthesis in a specific cell type within its native environment. Here we describe a method that enables the metabolic labeling, purification, and analysis of proteins in specific cell types and during defined periods in live animals. Using Caenorhabditis elegans, we show that labeling can be restricted to body wall muscles, intestinal epithelial cells, neurons, pharyngeal muscle, and cells that respond to heat shock. By coupling our methodology with isotopic labeling, we successfully identify proteins—including proteins with previously unknown expression patterns—expressed in targeted subsets of cells. Proteomic analysis of rare cells in heterogeneous environments presents difficult challenges. Systematic methods are needed to enrich, identify, and quantify proteins expressed in specific cells in complex biological systems including multicellular plants and animals. Here, we have engineered a Caenorhabditis elegans phenylalanyl-tRNA synthetase capable of tagging proteins with the reactive noncanonical amino acid p-azido-l-phenylalanine. We achieved spatiotemporal selectivity in the labeling of C. elegans proteins by controlling expression of the mutant synthetase using cell-selective (body wall muscles, intestinal epithelial cells, neurons, and pharyngeal muscle) or state-selective (heat-shock) promoters in several transgenic lines. Tagged proteins are distinguished from the rest of the protein pool through bioorthogonal conjugation of the azide side chain to probes that permit visualization and isolation of labeled proteins. By coupling our methodology with stable-isotope labeling of amino acids in cell culture (SILAC), we successfully profiled proteins expressed in pharyngeal muscle cells, and in the process, identified proteins not previously known to be expressed in these cells. Our results show that tagging proteins with spatiotemporal selectivity can be achieved in C. elegans and illustrate a convenient and effective approach for unbiased discovery of proteins expressed in targeted subsets of cells.

Identification of secreted bacterial proteins by noncanonical amino acid tagging

Significance Microbial pathogens use complex secretion systems to deliver virulence factors into host cells, where they disrupt host cell function. Understanding these systems is essential to the development of new treatments for infectious disease. A challenge in such studies arises from the abundance of host cell proteins, which interfere with detection of microbial effectors. Here we describe a metabolic labeling strategy that allows selective enrichment of microbial proteins from the host cell cytoplasm. The method enables efficient identification of microbial proteins that have been delivered to the host, identifies distinct secretion profiles for intracellular and extracellular bacteria, and allows for determination of the order of injection of microbial proteins into host cells. Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy.

Comprehensive proteomic profiling of outer membrane vesicles from Campylobacter jejuni

UNLABELLED Gram-negative bacteria constitutively release outer membrane vesicles (OMVs) during cell growth that play significant roles in bacterial survival, virulence and pathogenesis. In this study, comprehensive proteomic analysis of OMVs from a human gastrointestinal pathogen Campylobacter jejuni NCTC11168 was performed using high-resolution mass spectrometry. The OMVs of C. jejuni NCTC11168 were isolated from culture supernatants then characterized using electron microscopy and dynamic light scattering revealing spherical OMVs of an average diameter of 50nm. We then identified 134 vesicular proteins using high-resolution LTQ-Orbitrap mass spectrometry. Subsequent functional analysis of the genes revealed the relationships of the vesicular proteins. Furthermore, known N-glycoproteins were identified from the list of the vesicular proteome, implying the potential role of the OMVs as a delivery means for biologically relevant bacterial glycoproteins. These results enabled us to elucidate the overall proteome profile of pathogenic bacterium C. jejuni and to speculate on the function of OMVs in bacterial infections and communication. BIOLOGICAL SIGNIFICANCE This work demonstrates the importance of understanding vesicular proteomes from a human pathogen Campylobacter jejuni. From the secreted outer membrane vesicles (OMVs) of C. jejuni NCTC11168, we found a variety of virulence factors and essential proteins for bacterial survival. Bioinformatics analysis of these proteins predicted functional enrichment and localization. The most highly enriched were redox enzymes, which are considered to be essential for survival in oxygen-limiting environments and are predicted to be on the twin-arginine translocation (Tat) pathway suggesting a role for this pathway in the biogenesis of OMVs. This study additionally implicates a biological role for N-linked glycoproteins in OMVs. These approaches allow for a better understanding of the physiology of this important human pathogen.

Identification of host proteins associated with HIV-1 preintegration complexes isolated from infected CD4 + cells

An integrated HIV-1 genomic DNA leads to an infected cell becoming either an active or a latent virus-producing cell. Upon appropriate activation, a latently infected cell can result in production of progeny viruses that spread the infection to uninfected cells. The host proteins influence several steps of HIV-1 infection including formation of the preintegration complex (PIC), a key nucleoprotein intermediate essential for integration of reverse transcribed viral DNA into the chromosome. Much effort has gone into the identification of host proteins contributing to the assembly of functional PICs. Experimental approaches included the use of yeast two-hybrid system, co-immunoprecipitation, affinity tagged HIV-1 viral proteins and in vitro reconstitution of salt-stripped PIC activity. Several host proteins identified using these approaches have been shown to affect HIV-1 replication in cells and influence catalytic activities of recombinant IN in vitro. However, the comprehensive identification and characterization of host proteins associated with HIV-1 PICs of infected cells have been hindered in part by the technical limitation in acquiring sufficient amount of catalytically active PICs. To efficiently identify additional host factors associated with PICs in infected cells, we have developed the following novel approach. The catalytically active PICs from HIV-1-infected CD4+ cells were isolated using biotinylated target DNA, and the proteins selectively co-purifying with PICs have been analyzed by mass spectrometry. This technology enabled us to reveal at least 19 host proteins that are associated with HIV-1 PICs, of which 18 proteins have not been described previously with respect to HIV-1 integration. Physiological functions of the identified proteins range from chromatin organization to protein transport. A detailed characterization of these host proteins could provide new insights into the mechanism of HIV-1 integration and uncover new antiviral targets to block HIV-1 integration.

Phosphorylation of Synaptic GTPase Activating Protein (synGAP) by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) and Cyclin-dependent Kinase 5 (CDK5) Alters the Ratio of its GAP Activity Toward Ras and Rap GTPases

Background: synGAP inactivates Ras and Rap at synapses. Results: Phosphorylation of synGAP by CaMKII increases Rap1 GAP activity more than HRas GAP activity; phosphorylation by CDK5 has the opposite effect. Conclusion: Phosphorylation by CaMKII and CDK5 alters the ratio of Rap1 and HRas GAP activities. Significance: Phosphorylation of synGAP by CaMKII and CDK5 can alter the balance of synaptic functions regulated by Ras and Rap. synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAPs HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons.

Proteomics in the microbial sciences

Mass spectrometry based proteomics is now widely used in the microbial sciences. In conjunction with transcriptomics it has greatly enhanced the field of microbial biology and has provide microbiologists with unparalleled insights into cellular processes and functions. Proteomics allows the dynamic nature of the entire protein network to be mapped providing a deeper understanding of microbial systems, their evolution and role in disease states. This review is intended to provide an overview of mass spectrometry and its application to the field of microbial proteomics. Background is provided on the core mass analyzers, including the Orbitrap mass spectrometer, and novel fragmentation processes such as Electron Transfer Dissociation which leave post-translational modifications intact on peptide backbones allowing for their identification and localization. The review will also provide information on current key quantitative technologies and the state of the art in microbial metaproteomics.

Comprehensive profiling of N-linked glycosylation sites in HeLa cells using hydrazide enrichment.

The adenocarcinoma cell line HeLa serves as a model system for cancer research in general and cervical cancer in particular. In this study, hydrazide enrichment in combination with state-of-the art nanoLC-MS/MS analysis was used to profile N-linked glycosites in HeLa cells. N-Linked glycoproteins were selectively enriched in HeLa cells by the hydrazide capture method, which isolates all glycoproteins independent of their glycans. Nonglycosylated proteins were removed by extensive washing. N-Linked glycoproteins were identified with the specific NXT/S motif and deamidated asparagine (N). Deglycosylation was carried out in both H(2)(16)O and H(2)(18)O to confirm the deamidation. NanoLC-MS/MS analysis indicated that the method selectively enriched at least 100 fold N-linked glycosites in HeLa cells. When both the membrane and cytosolic fractions were used, a total of 268 unique N-glycosylation sites were identified corresponding to 106 glycoproteins. Bioinformatic analysis revealed that most of the glycoproteins identified are known to have an impact on cancer and have been proposed as biomarkers.

Protein Z: A putative novel biomarker for early detection of ovarian cancer

Ovarian cancer (OC) has the highest mortality of all gynaecological cancers. Early diagnosis offers an approach to achieving better outcomes. We conducted a blinded‐evaluation of prospectively collected preclinical serum from participants in the multimodal group of the United Kingdom Collaborative Trial of Ovarian Cancer Screening. Using isobaric tags (iTRAQ) we identified 90 proteins differentially expressed between OC cases and controls. A second targeted mass spectrometry analysis of twenty of these candidates identified Protein Z as a potential early detection biomarker for OC. This was further validated by ELISA analysis in 482 serial serum samples, from 80 individuals, 49 OC cases and 31 controls, spanning up to 7 years prior to diagnosis. Protein Z was significantly down‐regulated up to 2 years pre‐diagnosis (p = 0.000000411) in 8 of 19 Type I patients whilst in 5 Type II individuals, it was significantly up‐regulated up to 4 years before diagnosis (p = 0.01). ROC curve analysis for CA‐125 and CA‐125 combined with Protein Z showed a statistically significant (p= 0.00033) increase in the AUC from 77 to 81% for Type I and a statistically significant (p= 0.00003) increase in the AUC from 76 to 82% for Type II. Protein Z is a novel independent early detection biomarker for Type I and Type II ovarian cancer; which can discriminate between both types. Protein Z also adds to CA‐125 and potentially the Risk of Ovarian Cancer algorithm in the detection of both subtypes.

Mass Spectrometry in the Elucidation of the Glycoproteome of Bacterial Pathogens

Presently some three hundred post-translational modifications are known to occur in bacteria in vivo. Many of these modifications play critical roles in the regulation of proteins and control key biological processes. One of the most predominant modifications, N- and O-glycosylations are now known to be present in bacteria (and archaea) although they were long believed to be limited to eukaryotes. In a number of human pathogens these glycans have been found attached to the surfaces of pilin, flagellin and other surface and secreted proteins where it has been demonstrated that they play a role in the virulence of these bacteria. Mass spectrometry characterization of these glycosylation events has been the enabling key technology for these findings. This review will look at the use of mass spectrometry as a key technology for the detection and mapping of these modifications within microorganisms, with particular reference to the human pathogens, Campylobacter jejuni and Mycobacterium tuberculosis. The overall aim of this review will be to give a basic understanding of the current ‘state-of-the-art’ of the key techniques, principles and technologies, including bioinformatics tools, involved in the analysis of the glycosylation modifications.