Improving the Identification and Coverage of Plant Transmembrane Proteins in Medicago Using Bottom-Up Proteomics

Plant transmembrane proteins (TMPs) are essential for normal cellular homeostasis, nutrient exchange, and responses to environmental cues. Commonly used bottom-up proteomic approaches fail to identify a broad coverage of peptide fragments derived from TMPs. Here, we used mass spectrometry (MS) to compare the effectiveness of two solubilization and protein cleavage methods to identify shoot-derived TMPs from the legume Medicago. We compared a urea solubilization, trypsin Lys-C (UR-TLC) cleavage method to a formic acid solubilization, cyanogen bromide and trypsin Lys-C (FA-CTLC) cleavage method. We assessed the effectiveness of these methods by (i) comparing total protein identifications, (ii) determining how many TMPs were identified, and (iii) defining how many peptides incorporate all, or part, of transmembrane domains (TMD) sequences. The results show that the FA-CTLC method identified nine-fold more TMDs, and enriched more hydrophobic TMPs than the UR-TLC method. FA-CTLC identified more TMPs, particularly transporters, whereas UR-TLC preferentially identified TMPs with one TMD, particularly signaling proteins. The results suggest that combining plant membrane purification techniques with both the FA-CTLC and UR-TLC methods will achieve a more complete identification and coverage of TMPs.HL was supported by a Taiwan-ANU Scholarshi

First evidence of overlaps between HIV-Associated Dementia (HAD) and non-viral neurodegenerative diseases: proteomic analysis of the frontal cortex from HIV+ patients with and without dementia

<p>Abstract</p> <p>Background</p> <p>The pathogenesis of HIV-associated dementia (HAD) is poorly understood. To date, detailed proteomic fingerprinting directly from autopsied brain tissues of HAD and HIV non-dementia patients has not been performed.</p> <p>Result</p> <p>Here, we have analyzed total proteins from the frontal cortex of 9 HAD and 5 HIV non-dementia patients. Using 2-Dimensional differential in-gel electrophoresis (2-DIGE) to analyze the brain tissue proteome, 76 differentially expressed proteins (p < 0.05; fold change>1.25) were identified between HAD and HIV non-dementia patients, of which 36 protein spots (based on 3D appearance of spots on the images) were chosen for the mass spectrometry analysis. The large majority of identified proteins were represented in the energy metabolic (mitochondria) and signal transduction pathways. Furthermore, over 90% of the protein candidates are common to both HAD and other non-viral neurodegenerative disease, such as Alzheimer's disease. The data was further validated using specific antibodies to 4 proteins (CA2, GS, CKMT and CRMP2) by western blot (WB) in the same samples used for 2D-DIGE, with additional confirmation by immunohistochemitsry (IHC) using frontal lobe tissue from different HAD and HIV+ non-dementia patients. The validation for all 4 antibodies by WB and IHC was in concordance with the DIGE results, lending further credence to the current findings.</p> <p>Conclusion</p> <p>These results suggest not only convergent pathogenetic pathways for the two diseases but also the possibility of increased Alzheimer's disease (AD) susceptibility in HAD patients whose life expectancy has been significantly increased by highly active antiretroviral therapy.</p

Molecular characterization of the uncultivatable hemotropic bacterium Mycoplasma haemofelis

Mycoplasma haemofelis is a pathogenic feline hemoplasma. Despite its importance, little is known about its metabolic pathways or mechanism of pathogenicity due to it being uncultivatable. The recently sequenced M. haemofelis str. Langford 1 genome was analysed and compared to those of other available hemoplasma genomes

Diverse peptide hormones affecting root growth identified in the Medicago truncatula secreted peptidome

Multigene families encoding diverse secreted peptide hormones play important roles in plant development. A need exists to efficiently elucidate the structures and post-translational-modifications of these difficult-to-isolate peptide hormones in planta so that their biological functions can be determined. A mass spectrometry and bioinformatics approach was developed to comprehensively analyze the secreted peptidome of Medicago hairy root cultures and xylem sap. We identified 759 spectra corresponding to the secreted products of twelve peptide hormones including four CEP (C-TERMINALLY ENCODED PEPTIDE), two CLE (CLV3/ENDOSPERM SURROUNDING REGION RELATED) and six XAP (XYLEM SAP ASSOCIATED PEPTIDE) peptides. The MtCEP1, MtCEP2, MtCEP5 and MtCEP8 peptides identified differed in post-translational-modifications. Most were hydroxylated at conserved proline residues but some MtCEP1 derivatives were tri-arabinosylated. In addition, many CEP peptides possessed unexpected N- and C-terminal extensions. The pattern of these extensions suggested roles for endo- and exoproteases in CEP peptide maturation. Longer than expected, hydroxylated and homogeneously modified mono- and tri-arabinosylated CEP peptides corresponding to their in vivo structures were chemically synthesized to probe the effect of these post-translational-modifications on function. The ability of CEP peptides to elevate root nodule number was increased by hydroxylation at key positions. MtCEP1 peptides with N-terminal extensions or with tri-arabinosylation modification, however, were unable to impart increased nodulation. The MtCLE5 and MtCLE17 peptides identified were of precise size, and inhibited main root growth and increased lateral root number. Six XAP peptides, each beginning with a conserved DY sulfation motif, were identified including MtXAP1a, MtXAP1b, MtXAP1c, MtXAP3, MtXAP5 and MtXAP7. MtXAP1a and MtXAP5 inhibited lateral root emergence. Transcriptional analyses demonstrated peptide hormone gene expression in the root vasculature and tip. Since hairy roots can be induced on many plants, their corresponding root cultures may represent ideal source materials to efficiently identify diverse peptide hormones in vivo in a broad range of species.This work was supported by ARC grants to MAD: DP150104050 and LP150100826. NP was partly supported by an Endeavor Fellowship. NAMR was supported by an ANU Ph.D. scholarship supported by DP120101893. AI was supported by an Australian Post-graduate Award and an AW Howard Memorial Award. LC was supported by the Bruce-Veness Chandler and the John A. Lamberton research scholarship

Novel Approaches to Detect Serum Biomarkers for Clinical Response to Interferon-β Treatment in Multiple Sclerosis

Interferon beta (IFNβ) is the most common immunomodulatory treatment for relapsing-remitting multiple sclerosis (RRMS). However, some patients fail to respond to treatment. In this study, we identified putative clinical response markers in the serum and plasma of people with multiple sclerosis (MS) treated with IFNβ. In a discovery-driven approach, we use 2D-difference gel electrophoresis (DIGE) to identify putative clinical response markers and apply power calculations to identify the sample size required to further validate those markers. In the process we have optimized a DIGE protocol for plasma to obtain cost effective and high resolution gels for effective spot comparison. APOA1, A2M, and FIBB were identified as putative clinical response markers. Power calculations showed that the current DIGE experiment requires a minimum of 10 samples from each group to be confident of 1.5 fold difference at the p<0.05 significance level. In a complementary targeted approach, Cytometric Beadarray (CBA) analysis showed no significant difference in the serum concentration of IL-6, IL-8, MIG, Eotaxin, IP-10, MCP-1, and MIP-1α, between clinical responders and non-responders, despite the association of these proteins with IFNβ treatment in MS

The molecular details of a novel phosphorylation-dependent interaction between MRN and the SOSS complex

The repair of double-strand DNA breaks (DSBs) by homologous recombination is crucial in the maintenance of genome integrity. While the key role of the Mre11-Rad50-Nbs1 (MRN) complex in repair is well known, hSSB1 (SOSSB and OBFC2B), one of the main components of the sensor of single-stranded DNA (SOSS) protein complex, has also been shown to rapidly localize to DSB breaks and promote repair. We have previously demonstrated that hSSB1 binds directly to Nbs1, a component of the MRN complex, in a DNA damage-independent manner. However, recruitment of the MRN complex has also been demonstrated by an interaction between Integrator Complex Subunit 3 (INTS3; also known as SOSSA), another member of the SOSS complex, and Nbs1. In this study, we utilize a combined approach of in silico, biochemical, and functional experiments to uncover the molecular details of INTS3 binding to Nbs1. We demonstrate that the forkhead-associated domain of Nbs1 interacts with INTS3 via phosphorylation-dependent binding to INTS3 at Threonine 592, with contributions from Serine 590. Based on these data, we propose a model of MRN recruitment to a DSB via INTS3

Anatomy Arcade

Anatomy truly gets interactive via these collection of fun and engaging games, videos, and other multimedia excursions. The site was created by Ben Crossett, a science teacher in Australia. Several years ago, he decided to craft some new resources like &quot;Poke a Muscle&quot; and &quot;Whack-A-Bone&quot; that would be both entertaining and educational. Visitors can look at the Games By Body System to get started, or they can also look over the Games By Type area. The types of games here include jigsaw puzzles, crosswords, word searches, and the nice catch all Just For Fun. The activities are also made interesting by the mere fact that they contain catchy visuals and jaunty tunes. The site also includes a place where teachers can offer feedback and an area just for students

Site-specific glycosylation profile of influenza A (H1N1) hemagglutinin through tandem mass spectrometry

The study of influenza virus evolution in humans has revealed a significant role of glycosylation profile alterations in the viral glycoproteins – hemagglutinin (HA) and neuraminidase (NA), in the emergence of both seasonal and pandemic strains. Viral antigenic drift can modify the number and location of glycosylation sites, altering a wide range of biological activities and the antigenic properties of the strain. In view of the key role of glycans in determining antigenicity, elucidating the glycosylation profiles of influenza strains is a requirement towards the development of improved vaccines. Sequence-based analysis of viral RNA has provided great insight into the role of glycosite modifications in altering virulence and pathogenicity. Nonetheless, this sequence-based approach can only predict potential glycosylation sites. Due to experimental challenges, experimental confirmation of the occupation of predicted glycosylation sites has only been carried out for a few strains. Herein, we utilized HCD/CID-MS/MS tandem mass spectrometry to characterize the site-specific profile of HA of an egg-grown H1N1 reference strain (A/New Caledonia/20/1999). We confirmed experimentally the occupancy of glycosylation sites identified by primary sequence analysis and determined the heterogeneity of glycan structures. Four glycosylation sequons on the stalk region (N28, N40, N304 and N498) and four on the globular head (N71, N104, N142 and N177) of the protein are occupied. Our results revealed a broad glycan microheterogeneity, i.e., a great diversity of glycan compositions present on each glycosite. The present methodology can be applied to characterize other viruses, particularly different influenza strains, to better understand the impact of glycosylation on biological activities and aid the improvement of influenza vaccines