Shotgun Protein Profile of Human Adipose Tissue and Its Changes in Relation to Systemic Amyloidoses

In systemic amyloidosis, accumulation of misfolded proteins as extracellular amyloid fibrils in tissues causes severe organ dysfunction, but the molecular events of tissue damage related to amyloid deposition are still largely unknown. Through the use of the MudPIT proteomic approach, comprehensive protein profiles of human amyloid-affected adipose tissue from patients and its control (non-amyloid-affected) counterpart were acquired. Label-free comparison between patients and controls made it possible to highlight differences related to the presence of amyloid, by describing up- and down-represented proteins, connected into interacting networks. In particular, extracellular matrix (ECM), protein folding, lipid metabolism, and mitochondrial functions were among the most affected structural/functional pathways. The reported results, obtained with no a priori hypotheses, represent a significant step forward in the clarification of the molecular mechanisms involved in amyloidoses at tissue level and are the premise for understanding protein misfolding diseases

Dissecting <i>Escherichia coli</i> Outer Membrane Biogenesis Using Differential Proteomics

<div><p>The cell envelope of Gram-negative bacteria is a complex multi-layered structure comprising an inner cytoplasmic membrane and an additional asymmetric lipid bilayer, the outer membrane, which functions as a selective permeability barrier and is essential for viability. Lipopolysaccharide, an essential glycolipid located in the outer leaflet of the outer membrane, greatly contributes to the peculiar properties exhibited by the outer membrane. This complex molecule is transported to the cell surface by a molecular machine composed of seven essential proteins LptABCDEFG that form a transenvelope complex and function as a single device. While advances in understanding the mechanisms that govern the biogenesis of the cell envelope have been recently made, only few studies are available on how bacterial cells respond to severe envelope biogenesis defects on a global scale. Here we report the use of differential proteomics based on Multidimensional Protein Identification Technology (MudPIT) to investigate how <i>Escherichia coli</i> cells respond to a block of lipopolysaccharide transport to the outer membrane. We analysed the envelope proteome of a <i>lptC</i> conditional mutant grown under permissive and non permissive conditions and identified 123 proteins whose level is modulated upon LptC depletion. Most such proteins belong to pathways implicated in cell envelope biogenesis, peptidoglycan remodelling, cell division and protein folding. Overall these data contribute to our understanding on how <i>E. coli</i> cells respond to LPS transport defects to restore outer membrane functionality.</p></div

Envelope proteins exhibiting a significant variation upon LptC depletion: transport/assembly.

a<p>inferred from ecocyc.org.</p>b<p>NCBI accession number.</p>c<p>DAve value ranges from −2 and +2; positive value for DAve indicates that the protein is more abundant in LptC+ (FL905 grown with 0.2% arabinose); negative value for DAve indicates that the protein is more abundant in LptC-depleted (FL905 grown without arabinose).</p

Venn diagram of proteins distribution across strains and growth conditions analysed.

<p>Proteins are identified from total membrane samples. wt, (PS201); LptC⁺ (PS202, <i>araBp-lptC</i>) grown under permissive condition (0,2% arabinose); LptC-depleted, PS202 grown under non permissive condition (without arabinose).</p

Envelope proteins exhibiting a significant variation upon LptC depletion: cell envelope biogenesis.

a<p>inferred from ecocyc.org.</p>b<p>NCBI accession number.</p>c<p>DAve value ranges from −2 and +2; positive value for DAve indicat that the protein is more abundant in LptC+ (grown with 0.2% arabinose); negative value for DAve indicates that the protein is more abundant in LptC-depleted (grown without arabinose).</p

Envelope proteins exhibiting a significant variation upon LptC depletion: peptidoglycan synthesis/remodeling and cell division.

a<p>inferred from ecocyc.org.</p>b<p>NCBI accession number.</p>c<p>DAve value ranges from −2 and +2; positive value for DAve indicates that the protein is more abundant in LptC+ (grown with 0.2% arabinose); negative value for DAve indicates that the protein is more abundant in LptC-depleted (grown without arabinose).</p

LPS structure and transport in <i>Escherichia coli</i>.

<p>A) Chemical structure of LPS. O-antigen is indicated in parenthesis as it is not synthesized in <i>E. coli</i> K12 derivatives. B) LPS transport from IM to OM. The MsbA protein catalyzes LPS flipping across the IM that is then exported to the cell surface by the Lpt machinery.</p

AsmA protein abundance and subcellular localization.

<p>PS201 (<i>asmA-SPA lptC⁺</i>) and PS202 (<i>asmA-SPA araBp-lptC</i>) cells were grown with or without arabinose as indicated. Total membrane protein extracts prepared as described in Materials and Methods were analysed by immunoblotting (panel A) or fractionated by sucrose density gradient (panel B). A) 10 µg of total membrane proteins were loaded in each lane. 55-kDa protein was used as loading control. B) Fractions were collected from the top of the gradient and immunoblotted using antibodies recognizing the 55-kDa protein as IM marker, LamB as OM marker. α-Flag antibodies were used to detect AsmA-SPA protein. wt, PS201; LptC⁺, PS202 (<i>araBp-lptC</i>) grown under permissive condition (with 0,2% arabinose); LptC-depleted, PS202 grown under non permissive condition (without arabinose).</p

Analysis of <em>Pseudomonas aeruginosa</em> Cell Envelope Proteome by Capture of Surface-Exposed Proteins on Activated Magnetic Nanoparticles

<div><p>We report on specific magneto-capturing followed by Multidimensional Protein Identification Technology (MudPIT) for the analysis of surface-exposed proteins of intact cells of the bacterial opportunistic pathogen <em>Pseudomonas aeruginosa</em>. The magneto-separation of cell envelope fragments from the soluble cytoplasmic fraction allowed the MudPIT identification of the captured and neighboring proteins. Remarkably, we identified 63 proteins captured directly by nanoparticles and 67 proteins embedded in the cell envelope fragments. For a high number of proteins, our analysis strongly indicates either surface exposure or localization in an envelope district. The localization of most identified proteins was only predicted or totally unknown. This novel approach greatly improves the sensitivity and specificity of the previous methods, such as surface shaving with proteases that was also tested on <em>P. aeruginosa</em>. The magneto-capture procedure is simple, safe, and rapid, and appears to be well-suited for envelope studies in highly pathogenic bacteria.</p> </div

Validation of NPs as magneto-capture tools of envelope structures.

<p>(A) Scheme of treatment of <i>P. aeruginosa</i> intact cells with activated NPs. Before treatment with activated NPs (dark purple circles), cells were washed by the culture medium to remove extracellular proteins. After treatment for 5 min, NPs were inactivated (pink circles) and cells disrupted. NPs were magnetically recovered and washed thoroughly. NPs that interact with cell surface can establish covalent bonds with free -NH₂ moieties (e.g. those of lysine of exposed proteins, red dots) and, upon cell lysis, envelope fragments that stick to NPs (NP-Env) can be magneto-captured. (B) Scheme of treatment with inactive NPs. Before treatment with inactive NPs (pink circles), cells were washed by the culture medium to remove extracellular proteins. Upon treatment for 5 min, cells were disrupted. NPs were magnetically recovered and washed thoroughly. Inactive NPs can interact with cell surface but no covalent bonding occurs and thus envelope fragments are not magneto-captured. (C) Reactive and inactive NPs, that had been used to treat <i>P. aeruginosa</i> intact cells as illustrated in (A) and (B), respectively, were loaded onto SDS-PAGE to analyze protein contents. M: protein molecular weight marker. (D) Fluorescence emission spectra (λ_ex: 390 nm; λ_em: 400–550 nm) of: unreacted NPs (NPs); NP-Env; NP-Env extensively washed with SDS at 60°C (NP-Env+SDS); total membrane preparation (Membranes). All spectra were taken in the presence of the hydrophobic fluorescent probe 0.1 mM 1-anilinonaphthalene-8-sulfonate, tracking the presence of lipids. Note the overlapping spectra of NP-Env and Membranes.</p