The atheroma plaque secretome stimulates the mobilization of endothelial progenitor cells ex vivo.

Endothelial progenitor cells (EPCs) constitute a promising alternative in cardiovascular regenerative medicine due to their assigned role in angiogenesis and vascular repair. In response to injury, EPCs promote vascular remodeling by replacement of damaged endothelial cells and/or by secreting angiogenic factors over the damaged tissue. Nevertheless, such mechanisms need to be further characterized. In the current approach we have evaluated the initial response of early EPCs (eEPCs) from healthy individuals after direct contact with the factors released by carotid arteries complicated with atherosclerotic plaques (AP), in order to understand the mechanisms underlying the neovascularization and remodeling properties assigned to these cells. Herein, we found that the AP secretome stimulated eEPCs proliferation and mobilization ex vivo, and such increase was accompanied by augmented permeability, cell contraction and also an increase of cell-cell adhesion in association with raised vinculin levels. Furthermore, a comparative mass spectrometry analysis of control versus stimulated eEPCs revealed a differential expression of proteins in the AP treated cells, mostly involved in cell migration, proliferation and vascular remodeling. Some of these protein changes were also detected in the eEPCs isolated from atherosclerotic patients compared to eEPCs from healthy donors. We have shown, for the first time, that the AP released factors activate eEPCs ex vivo by inducing their mobilization together with the expression of vasculogenic related markers. The present approach could be taken as a ex vivo model to study the initial activation of vascular cells in atherosclerosis and also to evaluate strategies looking to potentiate the mobilization of EPCs prior to clinical applications.

Quantitative Map of Proteome Dynamics during Neuronal Differentiation.

Neuronal differentiation is a multistep process that shapes and re-shapes neurons by progressing through several typical stages, including axon outgrowth, dendritogenesis, and synapse formation. To systematically profile proteome dynamics throughout neuronal differentiation, we took cultured rat hippocampal neurons at different developmental stages and monitored changes in protein abundance using a combination of stable isotope labeling and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Almost one third of all 4,500 proteins quantified underwent a more than 2-fold expression change during neuronal differentiation, indicating extensive remodeling of the neuron proteome. To highlight the strength of our resource, we studied the neural-cell-adhesion molecule 1 (NCAM1) and found that it stimulates dendritic arbor development by promoting actin filament growth at the dendritic growth cone. We anticipate that our quantitative map of neuronal proteome dynamics is a rich resource for further analyses of the many identified proteins in various neurodevelopmental processes.

Extracellular IL-33 cytokine, but not endogenous nuclear IL-33, regulates protein expression in endothelial cells.

IL-33 is a nuclear cytokine from the IL-1 family that plays important roles in health and disease. Extracellular IL-33 activates a growing number of target cells, including group 2 innate lymphoid cells, mast cells and regulatory T cells, but it remains unclear whether intracellular nuclear IL-33 has additional functions in the nucleus. Here, we used a global proteomic approach based on high-resolution mass spectrometry to compare the extracellular and intracellular roles of IL-33 in primary human endothelial cells, a major source of IL-33 protein in human tissues. We found that exogenous extracellular IL-33 cytokine induced expression of a distinct set of proteins associated with inflammatory responses in endothelial cells. In contrast, knockdown of endogenous nuclear IL-33 expression using two independent RNA silencing strategies had no reproducible effect on the endothelial cell proteome. These results suggest that IL-33 acts as a cytokine but not as a nuclear factor regulating gene expression in endothelial cells.

Lysine conjugation properties in human IgGs studied by integrating high-resolution native mass spectrometry and bottom-up proteomics.

Antibody-drug conjugates (ADCs) are a novel class of biopharmaceuticals several of which are now being investigated in clinical studies. In ADCs, potent cytotoxic drugs are coupled via a linker to reactive residues in IgG monoclonal antibodies. Linkage to lysine residues in the IgGs, using N-hydroxysuccinimide ester based chemistry, is one of the possible options. To control drug load and specificity, proper knowledge is required about which lysine residues are most accessible and reactive. Here, we combine native MS and bottom-up proteomics to monitor the overall drug load and site-specific lysine reactivity, using N-hydroxysuccinimide-based tandem mass tags. High-resolution Orbitrap native MS enables us to monitor and quantify, due to the achieved baseline resolution, the sequential incorporation of up to 69 tandem mass tag molecules into human IgGs. Complementary, bottom-up proteomics facilitates the identification of some very reactive "hot-spot" conjugation sites. However, we also identify lysine residues that are highly resistant to chemical labeling. Our integrated approach gives insight into the conjugation properties of IgGs at both the intact protein and residue levels, providing fundamental information for controlling drug load and specificity in lysine-linked ADCs.

Simultaneous assessment of kinetic, site-specific, and structural aspects of enzymatic protein phosphorylation.

Protein phosphorylation is a widespread process forming the mechanistic basis of cellular signaling. Up to now, different aspects, for example, site-specificity, kinetics, role of co-factors, and structure-function relationships have been typically investigated by multiple techniques that are incompatible with one another. The approach introduced here maximizes the amount of information gained on protein (complex) phosphorylation while minimizing sample handling. Using high-resolution native mass spectrometry on intact protein (assemblies) up to 150 kDa we track the sequential incorporation of phosphate groups and map their localization by peptide LC-MS/MS. On two model systems, the protein kinase G and the interplay between Aurora kinase A and Bora, we demonstrate the simultaneous monitoring of various aspects of the phosphorylation process, namely the effect of different cofactors on PKG autophosphorylation and the interaction of AurA and Bora as both an enzyme-substrate pair and physical binding partners.

ELL, a novel TFIIH partner, is involved in transcription restart after DNA repair.

DNA lesions that block transcription may cause cell death even when repaired, if transcription does not restart to reestablish cellular metabolism. However, transcription resumption after individual DNA-lesion repair remains poorly described in mechanistic terms and its players are largely unknown. The general transcription factor II H (TFIIH) is a major actor of both nucleotide excision repair subpathways of which transcription-coupled repair highlights the interplay between DNA repair and transcription. Using an unbiased proteomic approach, we have identified the protein eleven-nineteen lysine-rich leukemia (ELL) as a TFIIH partner. Here we show that ELL is recruited to UV-damaged chromatin in a Cdk7- dependent manner (a component of the cyclin-dependent activating kinase subcomplex of TFIIH). We demonstrate that depletion of ELL strongly hinders RNA polymerase II (RNA Pol II) transcription resumption after lesion removal and DNA gap filling. Lack of ELL was also observed to increase RNA Pol II retention to the chromatin during this process. Identifying ELL as an essential player for RNA Pol II restart during cellular DNA damage response opens the way to obtaining a mechanistic description of transcription resumption after DNA repair.

Label-free quantification and shotgun analysis of complex proteomes by one-dimensional SDS-PAGE/NanoLC-MS: evaluation for the large scale analysis of inflammatory human endothelial cells.

To perform differential studies of complex protein mixtures, strategies for reproducible and accurate quantification are needed. Here, we evaluated a quantitative proteomic workflow based on nanoLC-MS/MS analysis on an LTQ-Orbitrap-VELOS mass spectrometer and label-free quantification using the MFPaQ software. In such label-free quantitative studies, a compromise has to be found between two requirements: repeatability of sample processing and MS measurements, allowing an accurate quantification, and high proteomic coverage of the sample, allowing quantification of minor species. The latter is generally achieved through sample fractionation, which may induce experimental bias during the label-free comparison of samples processed, and analyzed independently. In this work, we wanted to evaluate the performances of MS intensity-based label-free quantification when a complex protein sample is fractionated by one-dimensional SDS-PAGE. We first tested the efficiency of the analysis without protein fractionation and could achieve quite good quantitative repeatability in single-run analysis (median coefficient of variation of 5%, 99% proteins with coefficient of variation <48%). We show that sample fractionation by one-dimensional SDS-PAGE is associated with a moderate decrease of quantitative measurement repeatability while largely improving the depth of proteomic coverage. We then applied the method for a large scale proteomic study of the human endothelial cell response to inflammatory cytokines, such as TNFα, interferon γ, and IL1ß, which allowed us to finely decipher at the proteomic level the biological pathways involved in endothelial cell response to proinflammatory cytokines.

IL-33 is processed into mature bioactive forms by neutrophil elastase and cathepsin G.

Interleukin-33 (IL-33) (NF-HEV) is a chromatin-associated nuclear cytokine from the IL-1 family, which has been linked to important diseases, including asthma, rheumatoid arthritis, ulcerative colitis, and cardiovascular diseases. IL-33 signals through the ST2 receptor and drives cytokine production in type 2 innate lymphoid cells (ILCs) (natural helper cells, nuocytes), T-helper (Th)2 lymphocytes, mast cells, basophils, eosinophils, invariant natural killer T (iNKT), and natural killer (NK) cells. We and others recently reported that, unlike IL-1ß and IL-18, full-length IL-33 is biologically active independently of caspase-1 cleavage and that processing by caspases results in IL-33 inactivation. We suggested that IL-33, which is released upon cellular damage, may function as an endogenous danger signal or alarmin, similar to IL-1α or high-mobility group box 1 protein (HMGB1). Here, we investigated the possibility that IL-33 activity may be regulated by proteases released during inflammation. Using a combination of in vitro and in vivo approaches, we demonstrate that neutrophil serine proteases cathepsin G and elastase can cleave full-length human IL-33(1-270) and generate mature forms IL-33(95-270), IL-33(99-270), and IL-33(109-270). These forms are produced by activated human neutrophils ex vivo, are biologically active in vivo, and have a ~10-fold higher activity than full-length IL-33 in cellular assays. Murine IL-33 is also cleaved by neutrophil cathepsin G and elastase, and both full-length and cleaved endogenous IL-33 could be detected in the bronchoalveolar lavage fluid in an in vivo model of acute lung injury associated with neutrophil infiltration. We propose that the inflammatory microenvironment may exacerbate disease-associated functions of IL-33 through the generation of highly active mature forms.

The THAP-zinc finger protein THAP1 associates with coactivator HCF-1 and O-GlcNAc transferase: a link between DYT6 and DYT3 dystonias.

THAP1 is a sequence-specific DNA binding factor that regulates cell proliferation through modulation of target genes such as the cell cycle-specific gene RRM1. Mutations in the THAP1 DNA binding domain, an atypical zinc finger (THAP-zf), have recently been found to cause DYT6 dystonia, a neurological disease characterized by twisting movements and abnormal postures. In this study, we report that THAP1 shares sequence characteristics, in vivo expression patterns and protein partners with THAP3, another THAP-zf protein. Proteomic analyses identified HCF-1, a potent transcriptional coactivator and cell cycle regulator, and O-GlcNAc transferase (OGT), the enzyme that catalyzes the addition of O-GlcNAc, as major cellular partners of THAP3. THAP3 interacts with HCF-1 through a consensus HCF-1-binding motif (HBM), a motif that is also present in THAP1. Accordingly, THAP1 was found to bind HCF-1 in vitro and to associate with HCF-1 and OGT in vivo. THAP1 and THAP3 belong to a large family of HCF-1 binding factors since seven other members of the human THAP-zf protein family were identified, which harbor evolutionary conserved HBMs and bind to HCF-1. Chromatin immunoprecipitation (ChIP) assays and RNA interference experiments showed that endogenous THAP1 mediates the recruitment of HCF-1 to the RRM1 promoter during endothelial cell proliferation and that HCF-1 is essential for transcriptional activation of RRM1. Together, our findings suggest HCF-1 is an important cofactor for THAP1. Interestingly, our results also provide an unexpected link between DYT6 and DYT3 (X-linked dystonia-parkinsonism) dystonias because the gene encoding the THAP1/DYT6 protein partner OGT maps within the DYT3 critical region on Xq13.1.