Karin M. Green

Proteomic analysis of human meibomian gland secretions

Background/aim: Human tears contain hundreds of proteins that may exert a significant influence on tear film stability, ocular surface integrity, and visual function. The authors hypothesise that many of these proteins originate from the meibomian gland. This study’s aim was to begin to develop the proteomic methodology to permit the testing of their hypothesis. Methods: Meibomian gland secretions were collected from the lower eyelids of adult volunteers and placed in a chloroform-methanol mixture. Samples were partitioned in a biphasic system and non-lipid phase materials were reduced, alkylated, and trypsin digested to obtain peptides for protein identification. This peptide mixture was separated by µ-capillary reverse phase chromatography and the effluent examined by nano-electrospray MS and data dependent MS/MS. SEQUEST software was used to identify proteins from the MS/MS spectra. Results: The methodological approach to date has permitted the identification of more than 90 proteins in human meibomian gland secretions. Proteins include the α2-macroglobulin receptor, IgA α chain, farnesoid X activated receptor, interferon regulatory factor 3, lacritin precursor, lactotransferrin, lipocalin 1, lysozyme C precursor, potential phospholipid transporting ATPase IK, seven transmembrane helix receptor (also termed somatostatin receptor type 4), testes development related NYD-SP21 (also termed high affinity IgE receptor β subunit), and TrkC tyrosine kinase. Conclusions: These findings indicate that the meibomian gland secretes a number of proteins into the tear film. It is quite possible that these proteins contribute to the dynamics of the tear film in both health and disease.

BIOCHEMICAL CHARACTERIZATION OF MEMBRANE FRACTIONS IN MURINE SPERM: IDENTIFICATION OF THREE DISTINCT SUB-TYPES OF MEMBRANE RAFTS

Despite enormous interest in membrane raft micro‐domains, no studies in any cell type have defined the relative compositions of the raft fractions on the basis of their major components—sterols, phospholipids, and proteins—or additional raft‐associating lipids such as the ganglioside, GM1. Our previous localization data in live sperm showed that the plasma membrane overlying the acrosome represents a stabilized platform enriched in GM1 and sterols. These findings, along with the physiological requirement for sterol efflux for sperm to function, prompted us to characterize sperm membrane fractions biochemically. After confirming limitations of commonly used detergent‐based approaches, we utilized a non‐detergent‐based method, separating membrane fractions that were reproducibly distinct based on sterol, GM1, phospholipid, and protein compositions (both mass amounts and molar ratios). Based on fraction buoyancy and biochemical composition, we identified at least three highly reproducible sub‐types of membrane raft. Electron microscopy revealed that raft fractions were free of visible contaminants and were separated by buoyancy rather than morphology. Quantitative proteomic comparisons and fluorescence localization of lipids suggested that different organelles contributed differentially to individual raft sub‐types, but that multiple membrane micro‐domain sub‐types could exist within individual domains. This has important implications for scaffolding functions broadly associated with rafts. Most importantly, we show that the common practice of characterizing membrane domains as either “raft” or “non‐raft” oversimplifies the actual biochemical complexity of cellular membranes. J. Cell. Physiol. 218: 537–548, 2009.

Mass spectrometry analysis of HIV-1 Vif reveals an increase in ordered structure upon oligomerization in regions necessary for viral infectivity

HIV‐1 Vif, an accessory protein in the viral genome, performs an important role in viral pathogenesis by facilitating the degradation of APOBEC3G, an endogenous cellular inhibitor of HIV‐1 replication. In this study, intrinsically disordered regions are predicted in HIV‐1 Vif using sequence‐based algorithms. Intrinsic disorder may explain why traditional structure determination of HIV‐1 Vif has been elusive, making structure‐based drug design impossible. To characterize HIV‐1 Vifs structural topology and to map the domains involved in oligomerization we used chemical cross‐linking, proteolysis, and mass spectrometry. Cross‐linking showed evidence of monomer, dimer, and trimer species via denaturing gel analysis and an additional tetramer via western blot analysis. We identified 47 unique linear peptides and 24 (13 intramolecular; 11 intermolecular) noncontiguous, cross‐linked peptides, among the noncross‐linked monomer, cross‐linked monomer, cross‐linked dimer, and cross‐linked trimer samples. Almost complete peptide coverage of the N‐terminus is observed in all samples analyzed, however reduced peptide coverage in the C‐terminal region is observed in the dimer and trimer samples. These differences in peptide coverage or “protections” between dimer and trimer indicate specific differences in packing between the two oligomeric forms. Intramolecular cross‐links within the monomer suggest that the N‐terminus is likely folded into a compact domain, while the C‐terminus remains intrinsically disordered. Upon oligomerization, as evidenced by the intermolecular cross‐links, the C‐terminus of one Vif protein becomes ordered by wrapping back on the N‐terminal domain of another. In addition, the majority of the intramolecular cross‐links map to regions that have been previously reported to be necessary for viral infectivity. Thus, this data suggests HIV‐1 Vif is in a dynamic equilibrium between the various oligomers potentially allowing it to interact with other binding partners. Proteins 2007.

In vitro evidence that commercial influenza vaccines are not similar in their ability to activate human T cell responses.

We evaluated three commercial trivalent inactivated vaccines (TIVs) from the 2007-2008 season in terms of their ability to elicit in vitro T cell responses. T cell-mediated immunity may offer a more cross-reactive vaccine approach for the prevention of pandemic or epidemic influenza. Human cytotoxic T cell lines demonstrated differences in matrix protein 1 and nucleocapsid protein recognition of autologous target cells. Peripheral blood mononuclear cells stimulated with each of the TIVs showed statistically significant differences between the vaccines in the numbers of IFNgamma producing cells activated. These data suggest that TIV vaccines are not similar in their ability to activate human T cell responses.

Proteomics analysis of perilymph and cerebrospinal fluid in mouse

Proteins in perilymph may alter the delivery profile of implantable intracochlear drug delivery systems through biofouling. Knowledge of protein composition will help anticipate interactions with delivered agents.

Endogenous U2·U5·U6 snRNA complexes in S. pombe are intron lariat spliceosomes

Excision of introns from pre-mRNAs is mediated by the spliceosome, a multi-megadalton complex consisting of U1, U2, U4/U6, and U5 snRNPs plus scores of associated proteins. Spliceosome assembly and disassembly are highly dynamic processes involving multiple stable intermediates. In this study, we utilized a split TAP-tag approach for large-scale purification of an abundant endogenous U2·U5·U6 complex from Schizosaccharomyces pombe. RNAseq revealed this complex to largely contain excised introns, indicating that it is primarily ILS (intron lariat spliceosome) complexes. These endogenous ILS complexes are remarkably resistant to both high-salt and nuclease digestion. Mass spectrometry analysis identified 68, 45, and 43 proteins in low-salt-, high-salt-, and micrococcal nuclease-treated preps, respectively. The protein content of a S. pombe ILS complex strongly resembles that previously reported for human spliced product (P) and Saccharomyces cerevisiae ILS complexes assembled on single pre-mRNAs in vitro. However, the ATP-dependent RNA helicase Brr2 was either substoichiometric in low-salt preps or completely absent from high-salt and MNase preps. Because Brr2 facilitates spliceosome disassembly, its relative absence may explain why the ILS complex accumulates logarithmically growing cultures and the inability of S. pombe extracts to support in vitro splicing.

Serum-Induced Differentiation of Human Meibomian Gland Epithelial Cells

PURPOSE We hypothesize that culturing immortalized human meibomian gland epithelial cells in serum-containing medium will induce their differentiation. The purpose of this investigation was to begin to test our hypothesis, and explore the impact of serum on gene expression and lipid accumulation in human meibomian gland epithelial cells. METHODS Immortalized and primary human meibomian gland epithelial cells were cultured in the presence or absence of serum. Cells were evaluated for lysosome and lipid accumulation, polar and neutral lipid profiles, and gene expression. RESULTS Our results support our hypothesis that serum stimulates the differentiation of human meibomian gland epithelial cells. This serum-induced effect is associated with a significant increase in the expression of genes linked to cell differentiation, epithelium development, the endoplasmic reticulum, Golgi apparatus, vesicles, and lysosomes, and a significant decrease in gene activity related to the cell cycle, mitochondria, ribosomes, and translation. These cellular responses are accompanied by an accumulation of lipids within lysosomes, as well as alterations in the fatty acid content of polar and nonpolar lipids. Of particular importance, our results show that the molecular and biochemical changes of immortalized human meibomian gland epithelial cells during differentiation are analogous to those of primary cells. CONCLUSIONS Overall, our findings indicate that immortalized human meibomian gland epithelial cells may serve as an ideal preclinical model to identify factors that control cellular differentiation in the meibomian gland.

Vaccinia peptides eluted from HLA-DR1 isolated from virus- infected cells are recognized by CD4+ T cells from a vaccinated donor

Class II MHC proteins bind peptides and present them to CD4 (+) T cells as part of the immune systems surveillance of bodily tissues for foreign and pathogenic material. Antigen processing and presentation pathways have been characterized in detail in normal cells, but there is little known about the actual viral peptides that are presented to CD4 (+) T cells that signal infection. In this study, two-dimensional LC-MS/MS was used to identify vaccinia virus-derived peptides among the hundreds to thousands of peptide antigens bound to the human class II MHC protein HLA-DR1 on the surface of vaccinia virus-infected cells. The peptides, derived from the I6L, D6R, and A10L viral proteins, were 15 residues in length, bound efficiently to HLA-DR1 as synthetic peptides, and were recognized by vaccinia-specific CD4 (+) T cells obtained from an immunized donor.

Striatal Synaptosomes from Hdh140Q/140Q Knock-in Mice have Altered Protein Levels, Novel Sites of Methionine Oxidation, and Excess Glutamate Release after Stimulation

BACKGROUND Synaptic connections are disrupted in patients with Huntingtons disease (HD). Synaptosomes from postmortem brain are ideal for synaptic function studies because they are enriched in pre- and post-synaptic proteins important in vesicle fusion, vesicle release, and neurotransmitter receptor activation. OBJECTIVE To examine striatal synaptosomes from 3, 6 and 12 month old WT and Hdh140Q/140Q knock-in mice for levels of synaptic proteins, methionine oxidation, and glutamate release. METHODS We used Western blot analysis, glutamate release assays, and liquid chromatography tandem mass spectrometry (LC-MS/MS). RESULTS Striatal synaptosomes of 6 month old Hdh140Q/140Q mice had less DARPP32, syntaxin 1 and calmodulin compared to WT. Striatal synaptosomes of 12 month old Hdh140Q/140Q mice had lower levels of DARPP32, alpha actinin, HAP40, Na+/K+-ATPase, PSD95, SNAP-25, TrkA and VAMP1, VGlut1 and VGlut2, increased levels of VAMP2, and modifications in actin and calmodulin compared to WT. More glutamate released from vesicles of depolarized striatal synaptosomes of 6 month old Hdh140Q/140Q than from age matched WT mice but there was no difference in glutamate release in synaptosomes of 3 and 12 month old WT and Hdh140Q/140Q mice. LC-MS/MS of 6 month old Hdh140Q/140Q mice striatal synaptosomes revealed that about 4% of total proteins detected (>600 detected) had novel sites of methionine oxidation including proteins involved with vesicle fusion, trafficking, and neurotransmitter function (synaptophysin, synapsin 2, syntaxin 1, calmodulin, cytoplasmic actin 2, neurofilament, and tubulin). Altered protein levels and novel methionine oxidations were also seen in cortical synaptosomes of 12 month old Hdh140Q/140Q mice. CONCLUSIONS Findings provide support for early synaptic dysfunction in Hdh140Q/140Q knock-in mice arising from altered protein levels, oxidative damage, and impaired glutamate neurotransmission and suggest that study of synaptosomes could be of value for evaluating HD therapies.

Haptoglobin as an early serum biomarker of virus-induced autoimmune type 1 diabetes in biobreeding diabetes resistant and LEW1.WR1 rats

Proteomic profiling of serum is a powerful technique to identify differentially expressed proteins that can serve as biomarkers predictive of disease onset. In this study, we utilized two-dimensional (2D) gel analysis followed by matrix-assisted-laser desorption/ionization time-of-flight mass spectrometry analysis to identify putative serum biomarkers for autoimmune type 1 diabetes (T1D) in biobreeding diabetes resistant (BBDR) rats induced to express the disease. Treatment with toll-like receptor 3 ligand, polyinosinic:polycytidilic acid (pIC), plus infection with Kilham rat virus (KRV), a rat parvovirus, results in nearly 100% of young BBDR rats becoming diabetic within 11–21 d. Sera collected from prediabetic rats at early time points following treatment with pIC + KRV were analyzed by 2D gel electrophoresis and compared with sera from control rats treated with phosphate-buffered saline, pIC alone or pIC + H1, a non-diabetogenic parvovirus. None of the latter three control treatments precipitates T1D. 2D gel analysis revealed that haptoglobin, an acute phase and hemoglobin scavenger protein, was differentially expressed in the sera of rats treated with pIC + KRV relative to control groups. These results were confirmed by Western blot and enzyme-linked immunosorbent assay studies, which further validated haptoglobin levels as being differentially increased in the sera of pIC + KRV-treated rats relative to controls during the first week following infection. Early elevations in serum haptoglobin were also observed in LEW1.WR1 rats that became diabetic following infection with rat cytomegalovirus. The identification and validation of haptoglobin as a putative serum biomarker for autoimmune T1D in rats now affords us the opportunity to test the validity of this protein as a biomarker for human T1D, particularly in those situations where viral infection is believed to precede the onset of disease.