Experimental workflow for <i>E</i>. <i>ruminantium</i> subcellular fractionation and proteome characterization.

<p><b>OM</b>, outer membrane; <b>I</b>, inner membrane; <b>C</b>, cytoplasm.</p

Evaluation of OM isolation quality.

<p>Transmission electron microscopy of (A) purified <i>E</i>. <i>ruminantium</i> and (B) the insoluble precipitate after 0.1% sarkosyl treatment; scale bar = 200 nm. (C) SDS-PAGE and (D) Western blot of <b>E</b> (elementary bodies), <b>S</b> (sarkosyl-soluble fraction), and <b>OM</b> (outer membrane fraction) using monoclonal antibodies against Map1. <u><b>Band 1</b></u>: Map1-14, X5HG56, GroEL; <u><b>Band 2</b></u>: Map1+1, Map1, Map1-6, VirB10, VirB4, GroEL, PyrE, Q5HAR6, X5HG56, 30S-S8; <u><b>Band 3</b></u>: Map1+1, Map1-6, Map2, GroEL, PyrE, Q5HAR6, X5HG56, Q5FGC2, Q5HBI2, Q5FHJ9; <u><b>Band 4</b></u>: Map1, Map1-6, VirB4, GroEL, DnaK, BamA, FusA, Pnp, Q5HAR6, X5HG56, Q5FH07, Q5HBS6; <u><b>Band 5</b></u>: VirB4, VirB10, VirB11, DnaK, HtpG, GroEL, FusA, 30S-S1, Q5FGV5, Q93FS2; <u><b>Band 6</b></u>: Map1, Map1-14, VirB10, PleD, GroEL, DnaK, FtsZ, 30S-S1, Q5HB83, Q5FGA7, Q5HBE1; <u><b>Band 7</b></u>: Map1-14, GroEL, DnaK, FtsZ, HtpG; <u><b>Band 8</b></u>: Map1-6, Map1, GroEL, DnaK, FtsZ, BamA; <u><b>Band 9</b></u>: Map1-6, Map1, Map1+1, Map1-14, GroEL, DnaK, BamA, Q5FFE6, Q5HAR6; <u><b>Band 10</b></u>: Map1-11, Map1-13, Map1, Map1+1, Map1-6, VirB10, VirB9, Q5FFE6, Q5HAR6, Q5HBI2, Q5HA95; <u><b>Band 11</b></u>: Map1, Map2, BamA, DnaK, GroEL, FusA, Def, 50S-L4, PyrE, X5HG56, Q5HBI2; <u><b>Band 12</b></u>: 30S-S18, 30S-S12, 50S-L7/L12, 50S-L18, 50S-L24, 50S-L28 X5HG56, Q5HBN6; <u><b>Band 13</b></u>: HupB, X5HG56; <u><b>Band 14</b></u>: 30S-S12, 50S-L7/L12, 50S-L18, GroEL, YajC, PyrE.</p

Iminoboronates: A New Strategy for Reversible Protein Modification

Protein modification has entered the limelight of chemical and biological sciences, since, by appending small molecules into proteins surfaces, fundamental biological and biophysical processes may be studied and even modulated in a physiological context. Herein we present a new strategy to modify the lysine’s ε-amino group and the protein’s <i>N</i>-terminal, based on the formation of stable iminoboronates in aqueous media. This functionality enables the stable and complete modification of these amine groups, which can be reversible upon the addition of fructose, dopamine, or glutathione. A detailed DFT study is also presented to rationalize the observed stability toward hydrolysis of the iminoboronate constructs

Transthyretin Amyloidosis: Chaperone Concentration Changes and Increased Proteolysis in the Pathway to Disease

<div><p>Transthyretin amyloidosis is a conformational pathology characterized by the extracellular formation of amyloid deposits and the progressive impairment of the peripheral nervous system. Point mutations in this tetrameric plasma protein decrease its stability and are linked to disease onset and progression. Since non-mutated transthyretin also forms amyloid in systemic senile amyloidosis and some mutation bearers are asymptomatic throughout their lives, non-genetic factors must also be involved in transthyretin amyloidosis. We discovered, using a differential proteomics approach, that extracellular chaperones such as fibrinogen, clusterin, haptoglobin, alpha-1-anti-trypsin and 2-macroglobulin are overrepresented in transthyretin amyloidosis. Our data shows that a complex network of extracellular chaperones are over represented in human plasma and we speculate that they act synergistically to cope with amyloid prone proteins. Proteostasis may thus be as important as point mutations in transthyretin amyloidosis.</p></div

Proteome analysis of plasma from ATTR individuals.

<p>A– 2D-PAGE analysis of plasma proteins. Labeled spots show a statistically significant variation (p<0.05) and a minimal fold variation of 1.5. These spots were excised, tryptic digested and proteins identified by MS/MS analysis. Average normalized volumes and protein identifications are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125392#pone.0125392.t001" target="_blank">Table 1</a>. B–Principle component analysis (PCA) of the 2D results. Each data point in the PCA represents the global expression values for all spots with a significant ANOVA value (p<0.05). A separation between the control and the ATTR individuals is clearly observed. C- 2D image analysis of four protein spots and normalized volumes, shown as examples. D-Over expression of western blot analysis of plasma from four control and four FAP individuals to detect TTR. E—Western blot analysis of a 2DE of serum from four control and four FAP individuals to detect TTR with super imposition of spots identified as TTR in 2DE.</p

Plasma from ATTR individuals presents a higher proteolytic activity.

<p>A- Proteolytic activity of control and ATTR plasma measured by fluorescent protease assay kit. B–Sequence coverage obtained for three spots identified as albumin with decreased molecular weight.</p

Extracellular Chaperones are overrepresented in DLT individuals—SDS-PAGE analysis of plasma proteins from four individuals after orthotic liver transplantation (OLT) and four individuals after domino transplantation (DLT) and Western blot to detect TTR, Fibrinogen, Alpha 1 anti-trypsin (ITI), Vitamin D binding protein (VBP), alpha 2 microglobolin and C-reactive protein (CRP).

<p>Molecular weight markers are represented on the left hand of the figure and included in the western blots as blue markers.</p