Diego Chiappe

Phosphorylation at S87 is enhanced in synucleinopathies, inhibits α-synuclein oligomerization and influences synuclein-membrane interactions.

Increasing evidence suggests that phosphorylation may play an important role in the oligomerization, fibrillogenesis, Lewy body (LB) formation, and neurotoxicity of α-synuclein (α-syn) in Parkinson disease. Herein we demonstrate that α-syn is phosphorylated at S87 in vivo and within LBs. The levels of S87-P are increased in brains of transgenic (TG) models of synucleinopathies and human brains from Alzheimer disease (AD), LB disease (LBD), and multiple system atrophy (MSA) patients. Using antibodies against phosphorylated α-syn (S129-P and S87-P), a significant amount of immunoreactivity was detected in the membrane in the LBD, MSA, and AD cases but not in normal controls. In brain homogenates from diseased human brains and TG animals, the majority of S87-P α-syn was detected in the membrane fractions. A battery of biophysical methods were used to dissect the effect of S87 phosphorylation on the structure, aggregation, and membrane-binding properties of monomeric α-syn. These studies demonstrated that phosphorylation at S87 expands the structure of α-syn, increases its conformational flexibility, and blocks its fibrillization in vitro. Furthermore, phosphorylation at S87, but not S129, results in significant reduction of α-syn binding to membranes. Together, our findings provide novel mechanistic insight into the role of phosphorylation at S87 and S129 in the pathogenesis of synucleinopathies and potential roles of phosphorylation in α-syn normal biology.

A quantitative telomeric chromatin isolation protocol identifies different telomeric states

Telomere composition changes during tumourigenesis, aging and in telomere syndromes in a poorly defined manner. Here we develop a quantitative telomeric chromatin isolation protocol (QTIP) for human cells, in which chromatin is cross-linked, immunopurified and analysed by mass spectrometry. QTIP involves stable isotope labelling by amino acids in cell culture (SILAC) to compare and identify quantitative differences in telomere protein composition of cells from various states. With QTIP, we specifically enrich telomeric DNA and all shelterin components. We validate the method characterizing changes at dysfunctional telomeres, and identify and validate known, as well as novel telomere-associated polypeptides including all THO subunits, SMCHD1 and LRIF1. We apply QTIP to long and short telomeres and detect increased density of SMCHD1 and LRIF1 and increased association of the shelterins TRF1, TIN2, TPP1 and POT1 with long telomeres. Our results validate QTIP to study telomeric states during normal development and in disease.

Electron capture and transfer dissociation: Peptide structure analysis at different ion internal energy levels

We decoupled electron-transfer dissociation (ETD) and collision-induced dissociation of charge-reduced species (CRCID) events to probe the lifetimes of intermediate radical species in ETD-based ion trap tandem mass spectrometry of peptides. Short-lived intermediates formed upon electron transfer require less energy for product ion formation and appear in regular ETD mass spectra, whereas long-lived intermediates require additional vibrational energy and yield product ions as a function of CRCID amplitude. The observed dependencies complement the results obtained by double-resonance electron-capture dissociation (ECD) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ECD in a cryogenic ICR trap. Compared with ECD FT-ICR MS, ion trap MS offers lower precursor ion internal energy conditions, leading to more abundant charge-reduced radical intermediates and larger variation of product ion abundance as a function of vibrational post-activation amplitude. In many cases decoupled CRCID after ETD exhibits abundant radical c-type and even-electron z-type ions, in striking contrast to predominantly even-electron c-type and radical z-type ions in ECD FT-ICR MS and especially activated ion-ECD, thus providing a new insight into the fundamentals of ECD/ETD.

Dissecting the mechanisms of tissue transglutaminase-induced cross-linking of alpha-synuclein: implications for the pathogenesis of Parkinson disease.

Tissue transglutaminase (tTG) has been implicated in the pathogenesis of Parkinson disease (PD). However, exactly how tTG modulates the structural and functional properties of α-synuclein (α-syn) and contributes to the pathogenesis of PD remains unknown. Using site-directed mutagenesis combined with detailed biophysical and mass spectrometry analyses, we sought to identify the exact residues involved in tTG-catalyzed cross-linking of wild-type α-syn and α-syn mutants associated with PD. To better understand the structural consequences of each cross-linking reaction, we determined the effect of tTG-catalyzed cross-linking on the oligomerization, fibrillization, and membrane binding of α-syn in vitro. Our findings show that tTG-catalyzed cross-linking of monomeric α-syn involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed cross-linked monomeric α-syn composed of either wild-type or Gln → Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping by mass spectrometry. Using this approach, we identified the glutamine and lysine residues involved in tTG-catalyzed intramolecular cross-linking of α-syn. These studies demonstrate for the first time that Gln79 and Gln109 serve as the primary tTG reactive sites. Mutating both residues to asparagine abolishes tTG-catalyzed cross-linking of α-syn and tTG-induced inhibition of α-syn fibrillization in vitro. To further elucidate the sequence and structural basis underlying these effects, we identified the lysine residues that form isopeptide bonds with Gln79 and Gln109. This study provides mechanistic insight into the sequence and structural basis of the inhibitory effects of tTG on α-syn fibrillogenesis in vivo, and it sheds light on the potential role of tTG cross-linking on modulating the physiological and pathogenic properties of α-syn.Tissue transglutaminase (tTG) has been implicated in the pathogenesis of Parkinson disease (PD). However, exactly how tTG modulates the structural and functional properties of alpha-synuclein (alpha-syn) and contributes to the pathogenesis of PD remains unknown. Using site-directed mutagenesis combined with detailed biophysical and mass spectrometry analyses, we sought to identify the exact residues involved in tTG-catalyzed cross-linking of wild-type alpha-syn and alpha-syn mutants associated with PD. To better understand the structural consequences of each cross-linking reaction, we determined the effect of tTG-catalyzed cross-linking on the oligomerization, fibrillization, and membrane binding of alpha-syn in vitro. Our findings show that tTG-catalyzed cross-linking of monomeric alpha-syn involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed cross-linked monomeric alpha-syn composed of either wild-type or Gln --> Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping by mass spectrometry. Using this approach, we identified the glutamine and lysine residues involved in tTG-catalyzed intramolecular cross-linking of alpha-syn. These studies demonstrate for the first time that Gln(79) and Gln(109) serve as the primary tTG reactive sites. Mutating both residues to asparagine abolishes tTG-catalyzed cross-linking of alpha-syn and tTG-induced inhibition of alpha-syn fibrillization in vitro. To further elucidate the sequence and structural basis underlying these effects, we identified the lysine residues that form isopeptide bonds with Gln(79) and Gln(109). This study provides mechanistic insight into the sequence and structural basis of the inhibitory effects of tTG on alpha-syn fibrillogenesis in vivo, and it sheds light on the potential role of tTG cross-linking on modulating the physiological and pathogenic properties of alpha-syn.

Tissue transglutaminase mediated glutamine deamidation of beta-amyloid peptide increases peptide solubility, whereas enzymatic cross-linking and peptide fragmentation may serve as molecular triggers for rapid peptide aggregation.

Tissue transglutaminase (TGase) has been implicated in a number of cellular processes and disease states, where the enzymatic actions of TGase may serve in both, cell survival and apoptosis. To date, the precise functional properties of TGase in cell survival or cell death mechanisms still remain elusive. TGase-mediated cross-linking has been reported to account for the formation of insoluble lesions in conformational diseases. We report here that TGase induces intramolecular cross-linking of β-amyloid peptide (Aβ), resulting in structural changes of monomeric Aβ. Using high resolution mass spectrometry (MS) of cross-linked Aβ peptides, we observed a shift in mass, which is, presumably associated with the loss of NH3 due to enzymatic transamidation activity and hence intramolecular peptide cross-linking. We have observed that a large population of Aβ monomers contained an 0.984 Da increase in mass at a glutamine residue, indicating that glutamine 15 serves as an indispensable substrate in TGase-mediated deamidation to glutamate 15. We provide strong analytical evidence on TGase-mediated Aβ peptide dimerization, through covalent intermolecular cross-linking and hence the formation of Aβ1–40 dimers. Our in depth analyses indicate that TGase-induced post-translational modifications of Aβ peptide may serve as an important seed for aggregation.

Quantitative Mass Spectrometry Reveals Plasticity of Metabolic Networks in Mycobacterium smegmatis

Mycobacterium tuberculosis has a remarkable ability to persist within the human host as a clinically inapparent or chronically active infection. Fatty acids are thought to be an important carbon source used by the bacteria during long term infection. Catabolism of fatty acids requires reprogramming of metabolic networks, and enzymes central to this reprogramming have been targeted for drug discovery. Mycobacterium smegmatis, a nonpathogenic relative of M. tuberculosis, is often used as a model system because of the similarity of basic cellular processes in these two species. Here, we take a quantitative proteomics-based approach to achieve a global view of how the M. smegmatis metabolic network adjusts to utilization of fatty acids as a carbon source. Two-dimensional liquid chromatography and mass spectrometry of isotopically labeled proteins identified a total of 3,067 proteins with high confidence. This number corresponds to 44% of the predicted M. smegmatis proteome and includes most of the predicted metabolic enzymes. Compared with glucose-grown cells, 162 proteins showed differential abundance in acetate- or propionate-grown cells. Among these, acetate-grown cells showed a higher abundance of proteins that could constitute a functional glycerate pathway. Gene inactivation experiments confirmed that both the glyoxylate shunt and the glycerate pathway are operational in M. smegmatis. In addition to proteins with annotated functions, we demonstrate carbon source-dependent differential abundance of proteins that have not been functionally characterized. These proteins might play as-yet-unidentified roles in mycobacterial carbon metabolism. This study reveals several novel features of carbon assimilation in M. smegmatis, which suggests significant functional plasticity of metabolic networks in this organism.

Differential signaling networks of Bcr-Abl p210 and p190 kinases in leukemia cells defined by functional proteomics

The two major isoforms of the oncogenic Bcr–Abl tyrosine kinase, p210 and p190, are expressed upon the Philadelphia chromosome translocation. p210 is the hallmark of chronic myelogenous leukemia, whereas p190 occurs in the majority of B-cell acute lymphoblastic leukemia. Differences in protein interactions and activated signaling pathways that may be associated with the different diseases driven by p210 and p190 are unknown. We have performed a quantitative comparative proteomics study of p210 and p190. Strong differences in the interactome and tyrosine phosphoproteome were found and validated. Whereas the AP2 adaptor complex that regulates clathrin-mediated endocytosis interacts preferentially with p190, the phosphatase Sts1 is enriched with p210. Stronger activation of the Stat5 transcription factor and the Erk1/2 kinases is observed with p210, whereas Lyn kinase is activated by p190. Our findings provide a more coherent understanding of Bcr–Abl signaling, mechanisms of leukemic transformation, resulting disease pathobiology and responses to kinase inhibitors.

Characterization of the surfaceome of the metal-reducing bacterium Desulfotomaculum reducens

Desulfotomaculum reducens strain MI-1 is a Gram-positive, sulfate-reducing bacterium also capable of reducing Fe(III). Metal reduction in Gram-positive bacteria is poorly understood. Here, we investigated Fe(III) reduction with lactate, a non-fermentable substrate, as the electron donor. Lactate consumption is concomitant to Fe(III) reduction, but does not support significant growth, suggesting that little energy can be conserved from this process and that it may occur fortuitously. D. reducens can reduce both soluble [Fe(III)-citrate] and insoluble (hydrous ferric oxide, HFO) Fe(III). Because physically inaccessible HFO was not reduced, we concluded that reduction requires direct contact under these experimental conditions. This implies the presence of a surface exposed reductase capable of transferring electrons from the cell to the extracellular electron acceptor. With the goal of characterizing the role of surface proteins in D. reducens and of identifying candidate Fe(III) reductases, we carried out an investigation of the surface proteome (surfaceome) of D. reducens. Cell surface exposed proteins were extracted by trypsin cell shaving or by lysozyme treatment, and analyzed by liquid chromatography-tandem mass spectrometry. This investigation revealed that the surfaceome fulfills many functions, including solute transport, protein export, maturation and hydrolysis, peptidoglycan synthesis and modification, and chemotaxis. Furthermore, a few redox-active proteins were identified. Among these, three are putatively involved in Fe(III) reduction, i.e., a membrane-bound hydrogenase 4Fe-4S cluster subunit (Dred_0462), a heterodisulfide reductase subunit A (Dred_0143) and a protein annotated as alkyl hydroperoxide reductase but likely functioning as a thiol-disulfide oxidoreductase (Dred_1533).

Lysine acetylation stoichiometry and proteomics analyses reveal pathways regulated by sirtuin 1 in human cells.

Lysine acetylation is a widespread posttranslational modification affecting many biological pathways. Recent studies indicate that acetylated lysine residues mainly exhibit low acetylation occupancy, but challenges in sample preparation and analysis make it difficult to confidently assign these numbers, limiting understanding of their biological significance. Here, we tested three common sample preparation methods to determine their suitability for assessing acetylation stoichiometry in three human cell lines, identifying the acetylation occupancy in more than 1,300 proteins from each cell line. The stoichiometric analysis in combination with quantitative proteomics also enabled us to explore their functional roles. We found that higher abundance of the deacetylase sirtuin 1 (SIRT1) correlated with lower acetylation occupancy and lower levels of ribosomal proteins, including those involved in ribosome biogenesis and rRNA processing. Treatment with the SIRT1 inhibitor EX-527 confirmed SIRT1s role in the regulation of pre-rRNA synthesis and processing. Specifically, proteins involved in pre-rRNA transcription, including subunits of the polymerase I and SL1 complexes and the RNA polymerase I-specific transcription initiation factor RRN3, were up-regulated after SIRT1 inhibition. Moreover, many protein effectors and regulators of pre-rRNA processing needed for rRNA maturation were also up-regulated after EX-527 treatment with the outcome that pre-rRNA and 28S rRNA levels also increased. More generally, we found that SIRT1 inhibition down-regulates metabolic pathways, including glycolysis and pyruvate metabolism. Together, these results provide the largest data set thus far of lysine acetylation stoichiometry (available via ProteomeXchange with identifier PXD005903) and set the stage for further biological investigations of this central posttranslational modification.

Beyond Unpredictability: The Importance of Reproducibility in Understanding the Protein Corona of Nanoparticles

While it is well established that the surface of a nanoparticle plays a pivotal role for the protein corona, the vast number of proteins present in biological media render general conclusions about affinities between nanoparticle surfaces and proteins nontrivial. Recently published articles increasingly reveal differences between systems and an ever increasing number of influencing factors for the protein corona. In contrast, the present study posits that the reported differences may, at least in part, be due to poor experimental design, which leads to biased results. The present study investigates protein adsorption onto silica nanoparticles with different chemical groups on the surface by the statistical analysis of triplicate measurements as well as control measurements. We demonstrate that 60% of the identified protein types did not have any significant affinities for the nanoparticles. Of the remaining 40%, 60% were driven by surface charges and only 40% preferentially adsorbed onto specific surface groups. Furthermore, we found that of the 20 most abundant proteins in the serum, only five bound to the nanoparticles studied here. We illustrate the importance of control replicate experiments to avoid exaggerated differences between systems and to properly quantify the differences and similarities between comparable systems.