Shared and Unique Disease Pathways in Amyotrophic Lateral Sclerosis and Parkinson's Disease Unveiled in Peripheral Blood Mononuclear Cells.

Recent evidence supports an association between amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). Indeed, prospective population-based studies demonstrated that about one-third of ALS patients develop parkinsonian (PK) signs, even though different neuronal circuitries are involved. In this context, proteomics represents a valuable tool to identify unique and shared pathological pathways. Here, we used two-dimensional electrophoresis to obtain the proteomic profile of peripheral blood mononuclear cells (PBMCs) from PD and ALS patients including a small cohort of ALS patients with parkinsonian signs (ALS-PK). After the removal of protein spots correlating with confounding factors, we applied a sparse partial least square discriminant analysis followed by recursive feature elimination to obtain two protein classifiers able to discriminate (i) PD and ALS patients (30 spots) and (ii) ALS-PK patients among all ALS subjects (20 spots). Functionally, the glycolysis pathway was significantly overrepresented in the first signature, while extracellular interactions and intracellular signaling were enriched in the second signature. These results represent molecular evidence at the periphery for the classification of ALS-PK as ALS patients that manifest parkinsonian signs, rather than comorbid patients suffering from both ALS and PD. Moreover, we confirmed that low levels of fibrinogen in PBMCs is a characteristic feature of PD, also when compared with another movement disorder. Collectively, we provide evidence that peripheral protein signatures are a tool to differentially investigate neurodegenerative diseases and highlight altered biochemical pathways.

Proteomics turns functional.

Proteomics is acquiring a pivotal role in the comprehensive understanding of human biology. Biochemical processes involved in complex diseases, such as neurodegenerative diseases, diabetes and cancer, can be identified by combining proteomics analysis and bioinformatics tools. In the last ten years, the main output of differential proteomics investigations evolved from long lists of proteins to the generation of new hypotheses and their functional verification. The Journal of Proteomics participated to this progress, reporting more and more biologically-oriented papers with functional interpretation of proteomics data. This change in the field was due to both technological development and novel strategies in exploiting the deep characterization of proteomes. In this review, we explore several approaches that allow proteomics to turn functional. In particular, systems biology tools for data analysis are now routinely used to interpret results, thus defining the biological meaning of differentially abundant proteins. Moreover, by considering the importance of protein-protein interactions and the composition of macromolecular complexes, interactomics is complementing the information given by differential quantitative proteomics. Eventually, terminomics is unveiling new functions for cleaved proteoforms, by analyzing the effect of proteolysis globally. SIGNIFICANCE: Proteomics is rapidly evolving not only technologically but also strategically. The correct interpretation of proteomics data can reveal new functions of proteins in several biological backgrounds. Systems biology tools allow researchers to formulate new hypotheses to be further functionally tested. Interactomics is shedding new light on protein complexes truly involved in biochemical pathways and how their alteration can lead to dysfunctionality (in disease pathogenesis, for example). Terminomics is revealing the function of new discovered proteoforms and attributing a novel role to proteolysis. This review would provide the biologist important insights into current applications of several proteomic approaches that could offer new strategies to investigate biological systems.

Update of the Functional Mitochondrial Human Proteome Network.

Because of the pivotal role of mitochondrial alterations in several diseases, the Human Proteome Organization (HUPO) has promoted in recent years an initiative to characterize the mitochondrial human proteome, the mitochondrial human proteome project (mt-HPP). Here we generated an updated version of the functional mitochondrial human proteome network, made by nodes (mitochondrial proteins) and edges (gold binary interactions), using data retrieved from neXtProt, the reference database for HPP metrics. The principal new concept suggested was the consideration of mitochondria-associated proteins (first interactors), which may influence mitochondrial functions. All of the proteins described as mitochondrial in the sublocation or the GO Cellular Component sections of neXtProt were considered. Their other subcellular and submitochondrial localizations have been analyzed. The network represents the effort to collect all of the high-quality binary interactions described so far for mitochondrial proteins and the possibility for the community to reuse the information collected. As a proof of principle, we mapped proteins with no function, to speculate on their role by the background knowledge of their interactors, and proteins described to be involved in Parkinson's Disease, a neurodegenerative disorder, where it is known that mitochondria play a central role.

Network Analysis Identifies Disease-Specific Pathways for Parkinson's Disease.

Neurodegenerative diseases are characterized by the progressive loss of specific neurons in selected regions of the central nervous system. The main clinical manifestation (movement disorders, cognitive impairment, and/or psychiatric disturbances) depends on the neuron population being primarily affected. Parkinson's disease is a common movement disorder, whose etiology remains mostly unknown. Progressive loss of dopaminergic neurons in the substantia nigra causes an impairment of the motor control. Some of the pathogenetic mechanisms causing the progressive deterioration of these neurons are not specific for Parkinson's disease but are shared by other neurodegenerative diseases, like Alzheimer's disease and amyotrophic lateral sclerosis. Here, we performed a meta-analysis of the literature of all the quantitative proteomic investigations of neuronal alterations in different models of Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis to distinguish between general and Parkinson's disease-specific pattern of neurodegeneration. Then, we merged proteomics data with genetics information from the DisGeNET database. The comparison of gene and protein information allowed us to identify 25 proteins involved uniquely in Parkinson's disease and we verified the alteration of one of them, i.e., transaldolase 1 (TALDO1), in the substantia nigra of 5 patients. By using open-source bioinformatics tools, we identified the biological processes specifically affected in Parkinson's disease, i.e., proteolysis, mitochondrion organization, and mitophagy. Eventually, we highlighted four cellular component complexes mostly involved in the pathogenesis: the proteasome complex, the protein phosphatase 2A, the chaperonins CCT complex, and the complex III of the respiratory chain.

A systems biology-led insight into the role of the proteome in neurodegenerative diseases.

INTRODUCTION: Multifactorial disorders are the result of nonlinear interactions of several factors; therefore, a reductionist approach does not appear to be appropriate. Proteomics is a global approach that can be efficiently used to investigate pathogenetic mechanisms of neurodegenerative diseases. AREAS COVERED: Here, we report a general introduction about the systems biology approach and mechanistic insights recently obtained by over-representation analysis of proteomics data of cellular and animal models of Alzheimer's disease, Parkinson's disease and other neurodegenerative disorders, as well as of affected human tissues. Expert commentary: As an inductive method, proteomics is based on unbiased observations that further require validation of generated hypotheses. Pathway databases and over-representation analysis tools allow researchers to assign an expectation value to pathogenetic mechanisms linked to neurodegenerative diseases. The systems biology approach based on omics data may be the key to unravel the complex mechanisms underlying neurodegeneration.

Experimental setup for the identification of mitochondrial protease substrates by shotgun and top-down proteomics.

Mitochondria possess a proteolytic system that contributes to the regulation of mitochondrial dynamics, mitochondrial biogenesis and mitophagy. We aimed at the identification by bottom-up proteomics of altered protein processing due to the activation of mitochondrial proteases in a cellular model of impaired dopamine homeostasis. Moreover, we optimized the conditions for top-down proteomics to identify the cleavage site sequences.

Systems biology analysis of the proteomic alterations induced by MPP(+), a Parkinson's disease-related mitochondrial toxin.

Parkinson's disease (PD) is a complex neurodegenerative disease whose etiology has not been completely characterized. Many cellular processes have been proposed to play a role in the neuronal damage and loss: defects in the proteosomal activity, altered protein processing, increased reactive oxygen species burden. Among them, the involvement of a decreased activity and an altered disposal of mitochondria is becoming more and more evident. The mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP(+)), an inhibitor of complex I, has been widely used to reproduce biochemical alterations linked to PD in vitro and its precursor, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), to induce a Parkinson-like syndrome in vivo. Therefore, we performed a meta-analysis of the literature of all the proteomic investigations of neuronal alterations due to MPP(+) treatment and compared it with our results obtained with a mitochondrial proteomic analysis of SH-SY5Y cells treated with MPP(+). By using open-source bioinformatics tools, we identified the biochemical pathways and the molecular functions mostly affected by MPP(+), i.e., ATP production, the mitochondrial unfolded stress response, apoptosis, autophagy, and, most importantly, the synapse funcionality. Eventually, we generated protein networks, based on physical or functional interactions, to highlight the relationships among the molecular actors involved. In particular, we identified the mitochondrial protein HSP60 as the central hub in the protein-protein interaction network. As a whole, this analysis clarified the cellular responses to MPP(+), the specific mitochondrial proteome alterations induced and how this toxic model can recapitulate some pathogenetic events of PD.

Efficient resolution of racemic N-benzyl beta3-amino acids by iterative liquid-liquid extraction with a chiral (salen)cobalt(III) complex as enantioselective selector.

The efficient (up to 93% ee) resolution of racemic N-benzyl beta(3)-amino acids has been achieved by an iterative (two cycle) liquid-liquid extraction process using a lipophilic chiral (salen)cobalt(III) complex [Co(III)(1)(OAc)]. As a result of the resolution by extraction, one enantiomer of the N-benzyl beta(3)-amino acid predominated in the aqueous phase, while the other enantiomer was driven into the organic phase by complexation to cobalt. The complexed amino acid was then quantitatively released into an aqueous phase, by a reductive (Co(III)--> Co(II)) counter-extraction using l-ascorbic acid. The reductive cleavage allowed for the recovery of the cobalt(II) selector in up to 90% yield (easily re-oxidable to Co(III) with air/AcOH).

Rh-catalyzed enantioselective conjugate addition of arylboronic acids with a dynamic library of chiral tropos phosphorus ligands.

A library of 19 chiral tropos phosphorus ligands, based on a free-to-rotate (tropos) biphenol unit and a chiral P-bonded alcohol (11 phosphites, 1-P(O)(2)O to 11-P(O)(2)O) or secondary amine (8 phosphoramidites, 12-P(O)(2)N to 19-P(O)(2)N), were screened, individually and in combinations of two, in the rhodium-catalyzed asymmetric conjugate addition of arylboronic acids to enones and enoates. High enantioselectivities (up to 99 % ee) and excellent yields were obtained in the addition to either cyclic or acyclic substrates. The flexible biphenolic P ligands outperformed the analogous rigid binaphtholic P ligands. Variable-temperature (31)P NMR studies revealed that the biphenolic ligands are tropos even at low temperature. Only below 190 K was a coalescence observed; upon further cooling, two atropisomers were detected. The Rh homocomplexes ([Rh(L(a))(2)](+)) were also studied: in general, a single doublet (P-Rh coupling) was observed in the case of the biphenolic phosphite ligands, over the temperature range 380-230 K, demonstrating their tropos nature in the rhodium complexes even at low temperatures. On the other hand, the phosphoramidites showed different behaviors depending on the structure of the ligand and on the nature of the rhodium source. The spectrum at 230 K of the mixture of [Rh(acac)(eth)(2)] (eth=C(2)H(4)) with phosphite 6-P(O)(2)O and phosphoramidite 19-P(O)(2)N (the most enantioselective ligand combination in the conjugate addition reaction) revealed the presence of four homocomplexes (total approximately 40 %: [Rh{6-P(O)(2)O}(2)], [Rh{(aR)-19-P(O)(2)N}(2)], [Rh{(aS)-19-P(O)(2)N}(2)], [Rh{(aR)-19-P(O)(2)N}{(aS)-19-P(O)(2)N}]) and one heterocomplex, [Rh{6-P(O)(2)O}{(aR)-19-P(O)(2)N}] (approximately 60 %) In the heterocomplex, the biphenol-derived phosphite is free to rotate (tropos) while the biphenol-derived phosphoramidite shows a temperature-dependent tropos/atropos behavior (coalescence temperature=310 K).

A highly stereoselective synthesis of the C10-C23 fragment of (-)-dictyostatin.

A highly stereoselective synthesis of the C10-C23 fragment of (-)-dictyostatin has been achieved using a Carreira alkynylation and a Marshall-Tamaru allenylzinc addition as key steps.

Enantioselective conjugate addition of phenylboronic acid to enones catalysed by a chiral tropos/atropos rhodium complex at the coalescence temperature.

A highly enantioselective rhodium-catalysed conjugate addition of phenylboronic acid to cyclic enones has been achieved using a dynamic library of chiral phosphorus ligands; the tropos/atropos nature of the ligands in the rhodium complex has been characterised via 31P-NMR.

Rh-catalyzed asymmetric hydrogenation of prochiral olefins with a dynamic library of chiral TROPOS phosphorus ligands.

A library of 19 chiral tropos phosphorus ligands, based on a flexible (tropos) biphenol unit and a chiral P-bound alcohol (11 phosphites) or secondary amine (8 phosphoramidites), was synthesized. These ligands were screened, individually and as a combination of two, in the rhodium-catalyzed asymmetric hydrogenation of dehydro-alpha-amino acids, dehydro-beta-amino acids, enamides and dimethyl itaconate. ee values up to 98% were obtained for the dehydro-alpha-amino acids, by using the best combination of ligands, a phosphite [4-P(O)2O] and a phosphoramidite [13-P(O)2N]. Kinetic studies of the reactions with the single ligands and with the combination of phosphite [4-P(O)2O] and phosphoramidite [13-P(O)2N] have shown that the phosphite, despite being less enantioselective, promotes the hydrogenation of methyl 2-acetamidoacrylate and methyl 2-acetamidocinnamate faster than the mixture of the same phosphite with the phosphoramidite, while the phosphoramidite alone is much less active. In this way, the reaction was optimized by lowering the phosphite/phosphoramidite ratio (the best ratio is 0.25 equiv phosphite/1.75 equiv phosphoramidite) with a resulting improvement of the product enantiomeric excess. A simple mathematical model for a better understanding of the variation of the enantiomeric excess with the phosphite/phosphoramidite ratio is also presented.

The structure of the oligosaccharides of alpha3beta1 integrin from human ureter epithelium (HCV29) cell line.

There is a growing line of evidence that glycosylation of alpha and beta subunits is important for the function of integrins. Integrin alpha3beta1, from human ureter epithelium cell-line HCV29, was isolated by affinity chromatography on laminin GD6 peptide. Characterization of its carbohydrate moieties was carried out using sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by Western blotting on Immobilon P and on-blot deglycosylation with peptide N-glycosidase-F. Profiles of N-glycans for each subunit were obtained by matrix-assisted laser desorption/ionization mass spectrometry. Our findings demonstrated, in both subunits of integrin alpha3beta1, the presence of complex type oligosaccharides with a wide heterogeneity. Bi- tri- and tetraantennary structures were the most common, while high-mannose type structures were minor. Also the presence of short poly-N-acetyllactosamine entities was shown. These results show that while the predominant oligosaccharides of both subunits are identical, some slight differences between them do exist.