Pragya Singh

Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.

HDL lowers the risk for atherosclerotic cardiovascular disease by promoting cholesterol efflux from macrophage foam cells. However, other antiatherosclerotic properties of HDL are poorly understood. To test the hypothesis that the lipoprotein carries proteins that might have novel cardioprotective activities, we used shotgun proteomics to investigate the composition of HDL isolated from healthy subjects and subjects with coronary artery disease (CAD). Unexpectedly, our analytical strategy identified multiple complement-regulatory proteins and a diverse array of distinct serpins with serine-type endopeptidase inhibitor activity. Many acute-phase response proteins were also detected, supporting the proposal that HDL is of central importance in inflammation. Mass spectrometry and biochemical analyses demonstrated that HDL3 from subjects with CAD was selectively enriched in apoE, raising the possibility that HDL carries a unique cargo of proteins in humans with clinically significant cardiovascular disease. Collectively, our observations suggest that HDL plays previously unsuspected roles in regulating the complement system and protecting tissue from proteolysis and that the protein cargo of HDL contributes to its antiinflammatory and antiatherogenic properties.

A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria.

The functional spectrum of a secretion system is defined by its substrates. Here we analyzed the secretomes of Pseudomonas aeruginosa mutants altered in regulation of the Hcp Secretion Island-I-encoded type VI secretion system (H1-T6SS). We identified three substrates of this system, proteins Tse1-3 (type six exported 1-3), which are coregulated with the secretory apparatus and secreted under tight posttranslational control. The Tse2 protein was found to be the toxin component of a toxin-immunity system and to arrest the growth of prokaryotic and eukaryotic cells when expressed intracellularly. In contrast, secreted Tse2 had no effect on eukaryotic cells; however, it provided a major growth advantage for P. aeruginosa strains, relative to those lacking immunity, in a manner dependent on cell contact and the H1-T6SS. This demonstration that the T6SS targets a toxin to bacteria helps reconcile the structural and evolutionary relationship between the T6SS and the bacteriophage tail and spike.

Chemical Cross-Linking and Mass Spectrometry As a Low-Resolution Protein Structure Determination Technique

Protein complexes are the foundation of a majority of cellular processes. Although a large number of protein complexes have been identified through biochemical experiments, the precise molecular details and three-dimensional structures are available for only a small fraction. Chemical cross-linking coupled with mass spectrometry (CXMS) has gained popularity in recent years for characterization of inter- and intraprotein interactions in protein complexes. This perspective provides a comprehensive and critical overview of CXMS strategies employed for structural elucidation of protein complexes. We evaluate the challenges associated with CXMS techniques with special emphasis on data analysis. As sensitivity, mass resolution, mass accuracy and ease of use of mass spectrometers have improved, the complexity of processing and interpreting CXMS data has become the central problem to be addressed. We review here a number of computer programs available to address these problems.

xComb: A Cross-Linked Peptide Database Approach to Protein−Protein Interaction Analysis

We developed an informatic method to identify tandem mass spectra composed of chemically cross-linked peptides from those of linear peptides and to assign sequence to each of the two unique peptide sequences. For a given set of proteins the key software tool, xComb, combs through all theoretically feasible cross-linked peptides to create a database consisting of a subset of all combinations represented as peptide FASTA files. The xComb library of select theoretical cross-linked peptides may then be used as a database that is examined by a standard proteomic search engine to match tandem mass spectral data sets to identify cross-linked peptides. The database search may be conducted against as many as 50 proteins with a number of common proteomic search engines, e.g. Phenyx, Sequest, OMSSA, Mascot and X!Tandem. By searching against a peptide library of linearized, cross-linked peptides, rather than a linearized protein library, search times are decreased and the process is decoupled from any specific search engine. A further benefit of decoupling from the search engine is that protein cross-linking studies may be conducted with readily available informatics tools for which scoring routines already exist within the proteomic community.

VgrG-5 Is a Burkholderia Type VI Secretion System-Exported Protein Required for Multinucleated Giant Cell Formation and Virulence

ABSTRACT The type VI secretion system (T6SS) has emerged as a critical virulence factor for the group of closely related Burkholderia spp. that includes Burkholderia pseudomallei, B. mallei, and B. thailandensis. While the genomes of these bacteria, referred to as the Bptm group, appear to encode several T6SSs, we and others have shown that one of these, type VI secretion system 5 (T6SS-5), is required for virulence in mammalian infection models. Despite its pivotal role in the pathogenesis of the Bptm group, the effector repertoire of T6SS-5 has remained elusive. Here we used quantitative mass spectrometry to compare the secretome of wild-type B. thailandensis to that of a mutant harboring a nonfunctional T6SS-5. This analysis identified VgrG-5 as a novel secreted protein whose export depends on T6SS-5 function. Bioinformatics analysis revealed that VgrG-5 is a specialized VgrG protein that harbors a C-terminal domain (CTD) conserved among Bptm group species. We found that a vgrG-5 ΔCTD mutant is avirulent in mice and is unable to stimulate the fusion of host cells, a hallmark of the Bptm group previously shown to require T6SS-5 function. The singularity of VgrG-5 as a detected T6SS-5 substrate, taken together with the essentiality of its CTD for virulence, suggests that the protein is critical for the effector activity of T6SS-5. Intriguingly, we show that unlike the bacterial-cell-targeting T6SSs characterized so far, T6SS-5 localizes to the bacterial cell pole. We propose a model whereby the CTD of VgrG-5—, propelled by T6SS-5—, plays a key role in inducing membrane fusion, either by the recruitment of other factors or by direct participation.

Interactions between CusF and CusB identified by NMR spectroscopy and chemical cross-linking coupled to mass spectrometry

The Escherichia coli periplasmic proteins CusF and CusB, as part of the CusCFBA efflux system, aid in the resistance of elevated levels of copper and silver by direct metal transfer between the metallochaperone CusF and the membrane fusion protein CusB before metal extrusion from the periplasm to the extracellular space. Although previous in vitro experiments have demonstrated highly specific interactions between CusF and CusB that are crucial for metal transfer to occur, the structural details of the interaction have not been determined. Here, the interactions between CusF and CusB are mapped through nuclear magnetic resonance (NMR) spectroscopy and chemical cross-linking coupled with high-resolution mass spectrometry to better understand how recognition and metal transfer occur between these proteins. The NMR (1)H-(15)N correlation spectra reveal that CusB interacts with the metal-binding face of CusF. In vitro chemical cross-linking with a 7.7 Å homobifunctional amine-reactive cross-linker, BS(2)G, was used to capture the CusF/CusB interaction site, and mass spectral data acquired on an LTQ-Orbitrap confirm the following two cross-links: CusF K31 to CusB K29 and CusF K58 to CusB K32, thus revealing that the N-terminal region of CusB interacts with the metal-binding face of CusF. The proteins transiently interact in a metal-dependent fashion, and contacts between CusF and CusB are localized to regions near their respective metal-binding sites.

Detecting Cross-Linked Peptides by Searching against a Database of Cross-Linked Peptide Pairs

Mass spectrometric identification of cross-linked peptides can provide valuable information about the structure of protein complexes. We describe a straightforward database search scheme that identifies and assigns statistical confidence estimates to spectra from cross-linked peptides. The method is well suited to targeted analysis of a single protein complex, without requiring an isotope labeling strategy. Our approach uses a SEQUEST-style search procedure in which the database is comprised of a mixture of single peptides with and without linkers attached and cross-linked products. In contrast to several previous approaches, we generate theoretical spectra that account for all of the expected peaks from a cross-linked product, and we employ an empirical curve-fitting procedure to estimate statistical confidence measures. We show that our fully automated procedure successfully reidentifies spectra from a previous study, and we provide evidence that our statistical confidence estimates are accurate.

Interactions of the Transmembrane Polymeric Rings of the Salmonella enterica Serovar Typhimurium Type III Secretion System

ABSTRACT The type III secretion system (T3SS) is an interspecies protein transport machine that plays a major role in interactions of Gram-negative bacteria with animals and plants by delivering bacterial effector proteins into host cells. T3SSs span both membranes of Gram-negative bacteria by forming a structure of connected oligomeric rings termed the needle complex (NC). Here, the localization of subunits in the Salmonella enterica serovar Typhimurium T3SS NC were probed via mass spectrometry-assisted identification of chemical cross-links in intact NC preparations. Cross-links between amino acids near the amino terminus of the outer membrane ring component InvG and the carboxyl terminus of the inner membrane ring component PrgH and between the two inner membrane components PrgH and PrgK allowed for spatial localization of the three ring components within the electron density map structures of NCs. Mutational and biochemical analysis demonstrated that the amino terminus of InvG and the carboxyl terminus of PrgH play a critical role in the assembly and function of the T3SS apparatus. Analysis of an InvG mutant indicates that the structure of the InvG oligomer can affect the switching of the T3SS substrate to translocon and effector components. This study provides insights into how structural organization of needle complex base components promotes T3SS assembly and function. IMPORTANCE Many biological macromolecular complexes are composed of symmetrical homomers, which assemble into larger structures. Some complexes, such as secretion systems, span biological membranes, forming hydrophilic domains to move substrates across lipid bilayers. Type III secretion systems (T3SSs) deliver bacterial effector proteins directly to the host cell cytoplasm and allow for critical interactions between many Gram-negative pathogenic bacterial species and their hosts. Progress has been made towards the goal of determining the three-dimensional structure of the secretion apparatus by determination of high-resolution crystal structures of individual protein subunits and low-resolution models of the assembly, using reconstructions of cryoelectron microscopy images. However, a more refined picture of the localization of periplasmic ring structures within the assembly and their interactions has only recently begun to emerge. This work localizes T3SS transmembrane rings and identifies structural elements that affect substrate switching and are essential to the assembly of components that are inserted into host cell membranes. Many biological macromolecular complexes are composed of symmetrical homomers, which assemble into larger structures. Some complexes, such as secretion systems, span biological membranes, forming hydrophilic domains to move substrates across lipid bilayers. Type III secretion systems (T3SSs) deliver bacterial effector proteins directly to the host cell cytoplasm and allow for critical interactions between many Gram-negative pathogenic bacterial species and their hosts. Progress has been made towards the goal of determining the three-dimensional structure of the secretion apparatus by determination of high-resolution crystal structures of individual protein subunits and low-resolution models of the assembly, using reconstructions of cryoelectron microscopy images. However, a more refined picture of the localization of periplasmic ring structures within the assembly and their interactions has only recently begun to emerge. This work localizes T3SS transmembrane rings and identifies structural elements that affect substrate switching and are essential to the assembly of components that are inserted into host cell membranes.

An six-amino acid motif in the α3 domain of MICA is the cancer therapeutic target to inhibit shedding

Expression of the MHC class I chain related molecules A and B (MICA/B) on tumor cell surface can signal the immune receptor NKG2D for tumor immune destruction. However, MIC was found to be shed by tumors in cancer patients, which negatively regulates host immunity and promotes tumor immune evasion and progression. The mechanisms by which tumors shed MIC are not well understood although diverse groups of enzymes are suggested to be involved. The functional complexity of these enzymes makes them unfeasible therapeutic targets for inhibiting MIC shedding. Here we identified an six-amino acid (6-aa) motif in the alpha3 domain of MIC that is critical for the interaction of MIC with ERp5 to enable shedding. Mutations in this motif prevented MIC shedding but did not interfere with NKG2D-mediated recognition of MIC. Our study suggests that the 6-aa motif is a feasible target to inhibit MIC shedding for cancer therapy.

A pseudo-atomic model for the capsid shell of bacteriophage lambda using chemical cross-linking/mass spectrometry and molecular modeling.

Bacteriophage lambda is one of the most exhaustively studied of the double-stranded DNA viruses. Its assembly pathway is highly conserved among the herpesviruses and many of the bacteriophages, making it an excellent model system. Despite extensive genetic and biophysical characterization of many of the lambda proteins and the assembly pathways in which they are implicated, there is a relative dearth of structural information on many of the most critical proteins involved in lambda assembly and maturation, including that of the lambda major capsid protein. Toward this end, we have utilized a combination of chemical cross-linking/mass spectrometry and computational modeling to construct a pseudo-atomic model of the lambda major capsid protein as a monomer, as well as in the context of the assembled procapsid shell. The approach described here is generalizable and can be used to provide structural models for any biological complex of interest. The procapsid structural model is in good agreement with published biochemical data indicating that procapsid expansion exposes hydrophobic surface area and that this serves to nucleate assembly of capsid decoration protein, gpD. The model further implicates additional molecular interactions that may be critical to the assembly of the capsid shell and for the stabilization of the structure by the gpD decoration protein.