Histone Purification Combined with High-Resolution Mass Spectrometry to Examine Histone Post-Translational Modifications and Histone Variants in Caenorhabditis elegans

Histones are the major proteinaceous component of chromatin in eukaryotic cells and an important part of the epigenome, affecting most DNA-related events, including transcription, DNA replication, and chromosome segregation. The properties of histones are greatly influenced by their post-translational modifications (PTMs), over 200 of which are known today. Given this large number, researchers need sophisticated methods to study histone PTMs comprehensively. In particular, mass spectrometry (MS)-based approaches have gained popularity, allowing for the quantification of dozens of histone PTMs at once. Using these approaches, even the study of co-occurring PTMs and the discovery of novel PTMs become feasible. The success of MS-based approaches relies substantially on obtaining pure and well-preserved histones for analysis, which can be difficult depending on the source material. Caenorhabditis elegans has been a popular model organism to study the epigenome, but isolation of pure histones from these animals has been challenging. Here, we address this issue, presenting a method for efficient isolation of pure histone proteins from C. elegans at good yield. Further, we describe an MS pipeline optimized for accurate relative quantification of histone PTMs from C. elegans. We alkylate and tryptically digest the histones, analyze them by bottom-up MS, and then evaluate the resulting data by a C. elegans-adapted version of the software EpiProfile 2.0. Finally, we show the utility of this pipeline by determining differences in histone PTMs between C. elegans strains that age at different rates and thereby achieve very different lifespans. © 2020 The Authors. Basic Protocol 1: Large-scale growth and harvesting of synchronized C. elegans Basic Protocol 2: Nuclear preparation, histone extraction, and histone purification Basic Protocol 3: Bottom-up mass spectrometry analysis of histone PTMs and histone variants

DAF-16/FOXO requires Protein Phosphatase 4 to initiate transcription of stress resistance and longevity promoting genes

In C. elegans, the conserved transcription factor DAF-16/FOXO is a powerful aging regulator, relaying dire conditions into expression of stress resistance and longevity promoting genes. For some of these functions, including low insulin/IGF signaling (IIS), DAF-16 depends on the protein SMK-1/SMEK, but how SMK-1 exerts this role has remained unknown. We show that SMK-1 functions as part of a specific Protein Phosphatase 4 complex (PP4(SMK-1)). Loss of PP4(SMK-1) hinders transcriptional initiation at several DAF-16-activated genes, predominantly by impairing RNA polymerase II recruitment to their promoters. Search for the relevant substrate of PP4(SMK-1) by phosphoproteomics identified the conserved transcriptional regulator SPT-5/SUPT5H, whose knockdown phenocopies the loss of PP4(SMK-1). Phosphoregulation of SPT-5 is known to control transcriptional events such as elongation and termination. Here we also show that transcription initiating events are influenced by the phosphorylation status of SPT-5, particularly at DAF-16 target genes where transcriptional initiation appears rate limiting, rendering PP4(SMK-1) crucial for many of DAF-16's physiological roles

Expression proteomics study to determine metallodrug targets and optimal drug combinations

The emerging technique termed functional identification of target by expression proteomics (FITExP) has been shown to identify the key protein targets of anti-cancer drugs. Here, we use this approach to elucidate the proteins involved in the mechanism of action of two ruthenium(II)-based anti-cancer compounds, RAPTA-T and RAPTA-EA in breast cancer cells, revealing significant differences in the proteins upregulated. RAPTA-T causes upregulation of multiple proteins suggesting a broad mechanism of action involving suppression of both metastasis and tumorigenicity. RAPTA-EA bearing a GST inhibiting ethacrynic acid moiety, causes upregulation of mainly oxidative stress related proteins. The approach used in this work could be applied to the prediction of effective drug combinations to test in cancer chemotherapy clinical trials

Detection of viral proteins in human cells lines by xeno-proteomics: elimination of the last valid excuse for not testing every cellular proteome dataset for viral proteins.

Cell cultures used routinely in proteomic experiments may contain proteins from other species because of infection, transfection or just contamination. Since infection or contamination may affect the results of a biological experiment, it is important to test the samples for the presence of "alien" proteins. Usually cells are tested only for the most common infections, and most of the existing tests are targeting specific contaminations. Here we describe a three-step procedure for reliable untargeted detection of viral proteins using proteomics data, and recommend this or similar procedure to be applied to every proteomics dataset submitted for publication

Expression levels of the XMRV viral proteins within the proteome of the human cell line LNCaP.

<p>Small red dots correspond to the host cell proteins, whereas the two identified viral proteins are marked as cyan circles. Detected sequences of tryptic peptides of the two viral proteins are marked with color: red - peptides mapped perfectly on the XMRV sequence; blue and green - mutated sequences.</p

A non-radioactive mass spectrometry based differential radial capillary action of ligand assay (DRaCALA) to assess ligand binding to proteins

Binding of ligands to macromolecules changes their physicochemical characteristics. Cyclic di-GMP and other cyclic di-nucleotides are second messengers involved in motility/sessility and acute/chronic infection life style transition. Although the GGDEF domain encoding preferentially a diguanylate cyclase represents one of the most abundant bacterial domain superfamilies, the number of cyclic di-GMP receptors falls short. To facilitate screening for cyclic di-nucleotide binding proteins, we describe a non-radioactive, MALDI-TOF based modification of the widely applied differential radial capillary action of ligand assay (DRaCALA). The results of this assay suggest that YciRFec101, but not the YciRTOB1 variant of the diguanylate cyclase/phosphodiesterase YciR binds cyclic di-GMP

Changes in the plasma microvesicle proteome during the ovarian hyperstimulation phase of assisted reproductive technology

The incidence of pulmonary and venous thromboembolism is increased during the first trimester of pregnancies after assisted reproductive technology (ART) compared to spontaneous conception. We previously found that haemostatic plasma variables changed but within normal limits during controlled ovarian hyperstimulation (COH) concomitant with a major increase in plasma microvesicles (MVs) and markers indicating cell activation. We now explored the proteome of these MVs. Thirty-one women undergoing ART were blood sampled at down-regulation (DR) of oestrogen and at high level stimulation (HLS) with its 10-100-fold increased oestrogen level. Samples were analysed by liquid chromatography and tandem mass spectrometry to identify and quantify the proteome. We identified 306 proteins in the MVs and 72 had changed significantly at HLS compared to DR and more than 20% of them were associated with haemostasis. Thus, proteins related to both haemostasis and complement activation altered in plasma MVs in parallel with MV activation during COH. This needs to be further explored in the clinical context

Proteomic identification of heat shock induced danger signals in a melanoma cell lysate used in dendritic cell based cancer immunotherapy

Autologous dendritic cells (DCs) loaded with cancer cell-derived lysates have become a promising tool in cancer immunotherapy. During the last decade, we demonstrated that vaccination of advanced melanoma patients with autologous tumor antigen presenting cells (TAPCells) loaded with an allogeneic heat shock- (HS-) conditioned melanoma cell-derived lysate (called TRIMEL) is able to induce an antitumor immune response associated with a prolonged patient survival. TRIMEL provides not only a broad spectrum of potential melanoma-associated antigens but also danger signals that are crucial in the induction of a committed mature DC phenotype. However, potential changes induced by heat conditioning on the proteome of TRIMEL are still unknown. The identification of newly or differentially expressed proteins under defined stress conditions is relevant for understanding the lysate immunogenicity. Here, we characterized the proteomic profile of TRIMEL in response to HS treatment. A quantitative label-free proteome analysis of over 2800 proteins was performed, with 91 proteins that were found to be regulated by HS treatment: 18 proteins were overexpressed and 73 underexpressed. Additionally, 32 proteins were only identified in the HS-treated TRIMEL and 26 in non HS-conditioned samples. One protein from the overexpressed group and two proteins from the HS-exclusive group were previously described as potential damage-associated molecular patterns (DAMPs). Some of the HS-induced proteins, such as haptoglobin, could be also considered as DAMPs and candidates for further immunological analysis in the establishment of new putative danger signals with immunostimulatory functions.Chilean National Fund for Scientific and Technological Development FONDECYT 11130607 FONDEF ID16I10148 FONDECYT 117121

A Cyclic Di-GMP Network Is Present in Gram-Positive Streptococcus and Gram-Negative Proteus Species

Cyclic di-GMP is a ubiquitous second messenger in bacteria. This work describes the occurrence of a cyclic di-GMP signaling network in Gram-positive Streptococcus species and Gram-negative Proteus. After identification of candidate diguanylate cyclases by homology search in the respective species, the open reading frames were cloned and proteins expressed. Production of cyclic di-GMP was demonstrated by riboswitch assays, detection of cyclic di-GMP in cell lysates by MALDI-FTMS and in cell extracts by standard LC-MS/MS. Expression of the diguanylate cyclases in the heterologous host Salmonella typhimurium showed the expected physiological activity, namely up regulation of biofilm formation and down regulation of motility. The co-localisation of both sole diguanylate cyclases with cellulose or cellulose-like synthases indicates exopolysaccharide biosynthesis to be a conserved trait of cyclic di-GMP signaling