An optimized proteomics approach reveals novel alternative proteins in mouse liver development
Ontology highlight
ABSTRACT: Alternative open reading frames (AltORFs) are unannotated sequences in genome that encode novel peptides or proteins named alternative proteins (AltProts). Although ribosome profiling and bioinformatics predict a large number of AltProts, mass spectrometry (MS) as the only direct way of identification, is hampered by the short lengths and relative low abundance of AltProts. Here we report an approach based on size-exclusion chromatography (SEC) for simultaneous enrichment and fractionation of AltProts from mouse livers. With this strategy we compared and discovered 89 novel AltProts between embryonic and adult mouse livers and will extend our optimized workflow in various disease models in future study.
Project description:Proteogenomics is becoming a powerful tool in personalized medicine by linking genomics, transcriptomics and mass spectrometry (MS)-based proteomics. Due to increasing evidence of alternative open reading frame-encoded proteins (AltProts), proteogenomics has a high potential to unravel the characteristics, variants and expression levels of the alternative proteome, in addition to already annotated proteins (RefProts). To obtain a broader view of the proteome of ovarian cancer cells compared to ovarian epithelial cells, cell-specific total RNA-sequencing profiles and customized protein databases were generated. In total, 128 RefProts and 30 AltProts were identified exclusively in SKOV-3 and PEO-4 cells. Among them, an AltProt variant of IP_715944, translated from DHX8, was found mutated (p.Leu44Pro). We show high variation in protein expression levels of RefProts and AltProts in different subcellular compartments. The presence of 117 RefProt and two AltProt variants was described, along with their possible implications in the different physiological/pathological characteristics. To identify the possible involvement of AltProts in cellular processes, crosslinking-MS (XL-MS) was performed in each cell line to identify AltProt-RefProt interactions. This approach revealed an interaction between POLD3 and the AltProt IP_183088, which after molecular docking, was placed between POLD3-POLD2 binding sites, highlighting its possibility of the involvement in DNA replication and repair.
Project description:In our previous work, our investigation on macrophages has allowed us to show that the inhibition of the enzyme proprotein convertase (PC1/3) controls the activation of macrophages. We demonstrated that PC1/3 knockdown (KD) in macrophages would exhibit an increased secretion of pro-inflammatory and anti-tumoral factors. In this biological context, we assessed for histone modifications and for the presence and contribution of a “Ghost proteome” in these macrophages. In addition, we have identified a set of alternative proteins (AltProts) that have a key role in regulating various signaling pathways. In this study, to further investigate the underlying mechanisms involved in resistance of PC1/3 KD macrophages to anti-inflammatory stimuli, we have conducted a proteomics-systems biology study to assess the epigenome variation focusing on histone modifications and to investigate for the potential contribution of AltProts in regulating PC1/3 KD macrophages resistance. Results from our study have indicated the presence of significant variations in histone modifications along with the identification of 28 AltProts with in part involved in anti-tumoral resistance under IL-10 stimulation. These findings highlight that key role of altered epigenome histone modifications in driving resistance and indicates that, like the reference proteins, AltProts have major impact in the field of epigenetics and regulation of gene expression as shown in our results
Project description:Eukaryotic mRNA has long been considered monocistronic, but nowadays, alternative proteins (AltProts) challenge this tenet. The alternative or ghost proteome has largely been neglected and the involvement of AltProts in biological processes. Here, we used subcellular fractionation to increase the information about AltProts and facilitate the detection of protein-protein interactions by the identification of crosslinked peptides. In total, 112 unique AltProts were identified, and we were able to identify 220 crosslinks without peptide enrichment. Among these, 16 crosslinks between AltProts and Referenced Proteins (RefProts) were identified. We further focused on specific examples such as the interaction between IP_2292176 (AltFAM227B) and HLA-B, in which this protein could be a potential new immunopeptide, and the interactions between HIST1H4F and several AltProts which can play a role in mRNA transcription. Thanks to the study of the interactome and the localization of AltProts, we can reveal more of the importance of the ghost proteome.
Project description:Iron sulfur clusters (ISC) are essential cofactors that participate in electron transfer, environment sensing, and catalysis. Amongst the most ancient ISC containing proteins are the ferredoxin family of electron carriers. Humans have two ferredoxins, FDX1 and FDX2, localized to the mitochondria and important for ISC synthesis itself. We previously showed that hypoxia can bypass the requirement for some, but not all, components of the ISC biosynthetic pathway, but ferredoxins were not tested at that time. Here we report that FDX1 and its reductase FDXR, but not FDX2, are dispensable under ambient 1% O2 in cultured cells. We find that FDX1 is essential for production of the lipoic acid cofactor, which is synthesized by the ISC containing enzyme lipoyl synthase (LIAS). While hypoxia can rescue the growth phenotype of either FDX1 or LIAS knockout cells, lipoylation is not rescued, arguing against an alternative biosynthetic route or salvage pathway for lipoate in hypoxia. Our work identifies a role for FDX1/LIAS in lipoate synthesis and surprisingly reveals dispensability of lipoic acid altogether under low oxygen tensions in cell culture.
Project description:Large scale proteomic strategies rely on database interrogation. Thus, only referenced proteins can be identified. Recently, Alternative Proteins (AltProts) translated from nonannotated Alternative Open reading frame (AltORFs) were discovered using customized databases. Because of their small size which confers them peptide-like physicochemical properties, they are more difficult to detect using standard proteomics strategies. In this study, we tested different preparation workflows for improving the identification of AltProts in NCH82 human glioma cell line. The highest number of identified AltProts was achieved with RIPA buffer or boiling water extraction followed by acetic acid precipitation.
Project description:Meningiomas are the most common primary intracranial tumors and are associated with inactivation of the tumor suppressor NF2/Merlin, but one-third of meningiomas retain Merlin expression and typically have favorable clinical outcomes. Biochemical mechanisms underlying Merlin-intact meningioma growth are incompletely understood, and non-invasive biomarkers that predict meningioma outcomes and could be used to guide treatment de-escalation or imaging surveillance of Merlin-intact meningiomas are lacking. Here we integrate single-cell RNA sequencing, proximity-labeling proteomic mass spectrometry, mechanistic and functional approaches, and magnetic resonance imaging (MRI) across meningioma cells, xenografts, and human patients to define biochemical mechanisms and an imaging biomarker that distinguish Merlin-intact meningiomas with favorable clinical outcomes from meningiomas with unfavorable clinical outcomes. We find Merlin drives meningioma Wnt signaling and tumor growth through a feed-forward mechanism that requires Merlin dephosphorylation on serine 13 (S13) to attenuate inhibitory interactions with β-catenin and activate the Wnt pathway. Meningioma MRI analyses of xenografts and human patients show Merlin-intact meningiomas with S13 phosphorylation and favorable clinical outcomes are associated with high apparent diffusion coefficient (ADC) on diffusionweighted imaging. In sum, our results shed light on Merlin posttranslational modifications that regulate meningioma Wnt signaling and tumor growth in tumors without NF2/Merlin inactivation. To translate these findings to clinical practice, we establish a non-invasive imaging biomarker that could be used to guide treatment de-escalation or imaging surveillance for patients with favorable meningiomas.
Project description:Amyloidogenic proteins, characterized by their ability to form fibrillar aggregates with β sheet structure play an important role in several degenerative diseases, including Parkinson’s disease. Numerous amyloidogenic proteins, such as α synuclein, are intrinsically disordered (or contain ID regions, hence they also present as highly dynamic conformational ensembles in these regions. Aggregation is an inherent property of the polypeptide chains and under non physiological, appropriate conditions most of the proteins can aggregate and form polymers of various structures. Since amyloid fib ril s and oligomers are associated with a great variety of human diseases, inhibition of protein aggregation has great importance and it can be addressed by using small molecules, peptides or proteins. Our research aim was to study the potential application of modified forms of different amyloidogenic proteins as inhibitor molecules by introducing structural constraints in them. Under various conditions, different amyloidogenic proteins’ (α-synuclein and β2-microglobulin) monomer molecules have been cross linked intramolecularly and the heterogeneous mixtures has been fractionated by HPLC. The inhibitory potential of the isolated and effective molecules has been experimentally investigated by various methods ThT assay, TEM, CD spectroscopy). Furthermore, the locations of the crosslinks in the molecules were determined by mass spectrometry, and their structures are modeled in silico. Our results revealed that conformational constrains applied by cross linking on amyloidogenic proteins block their amyloid formation. Moreover, these molecules exhibited inhibitory effect on the aggregation of the unmodified proteins, as well.
Project description:Many proteins are lacking from reference databases because they are issued from alternative ORFs neither respecting the rules decreed for the genome annotation or for the protein translation from mRNA. It was evidenced that not only a single protein is therefore translated from an mRNA. However, the function of these Alternative Proteins (AltProts) remain largely unknown. Here, we were interested by looking to the function of AltProts in the context of cancer cell reprogramming. We have developed a large scale approach based on shot-gun proteomics and cross-linking mass spectrometry (XL-MS) to identify the regulation of the reference proteins (RefProts) versus the AltProts and find the their interaction partners. The study was conducted from NCH82 human glioma cells which were stimulated by the protein kinase A (PKA) activator Forskolin upon 16H, 24H and 48H to induce cell differentiation and epithelial-mesenchymal transition (EMT). The data have shown to enable tracing back the function of the AltProts by combining experimental data to in silico analysis using cytoscape with ClueGo for GO Term annotation and enrichment of pathways with String. Very interestingly many AltProts demonstrate to be involved in the regulation of tRNA through their interaction with aaRS proteins and of the cellular mobility.
Project description:Transcription-coupled DNA repair (TCR) efficiently removes bulky DNA lesions from transcribed parts of the genome and constitutes a major defence against DNA damage by UV irradiation. TCR begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CsB, the E3 ubiquitin ligase complex CRL4CsA, and the UV-stimulated scaffold protein A (UVSSA). Here we provide five high-resolution structures of Pol II transcription complexes with bound TCR factors and complementary biochemical data. Together with published work, our results converge on a model for the molecular mechanism of transcription-DNA repair coupling. In this model, Pol II stalling triggers the formation of an alternative transcription elongation complex that we call ECact. In ECact, CsB has replaced the elongation factor DSIF, binds the PAF1 complex, and repositions upstream DNA such that it contacts the elongation factor SPT6. The CsB translocase now pulls on the upstream DNA template, thereby pushing Pol II forward. If the lesion cannot be bypassed, Pol II gets stably stalled. CRL4CsA spans over the Pol II clamp to reach the Pol II jaw and direct ubiquitination of RPB1 residue lysine-1268. This triggers the recruitment of TFIIH to UVSSA, which is located at downstream DNA, and enables nucleotide excision repair. Finally, large-scale conformational changes in CRL4CsA enable ubiquitination of CsB and the release of TCR factors, thereby allowing Pol II to resume elongation and continue transcription over repaired DNA.
Project description:Plasma is a highly valuable resource for biomarker research since it is easy obtainable and contains a high amount of information on patient health status. Although advancements in the field of proteomics enabled analysis of the plasma proteome, identification of low abundant proteins remains challenging due to high complexity and large dynamic range. In order to reduce the dynamic range of protein concentrations, a tandem depletion technique consisting of ammonium sulfate precipitation and Protein A affinity chromatography was developed. Using this method, 50 % of albumin, together with other high abundant proteins such as alpha-1-antitrypin, was depleted from the plasma sample at 20 % to 40 % ammonium sulfate saturation levels. In combination with immunoglobulin removal using a Protein A column, this technique delivered up to 40 new low- to medium abundance protein identifications when performing a shotgun mass spectrometry analysis. Compared to non-depleted plasma, 270 additional protein spots were observed during 2D-PAGE analysis. These results illustrate that this tandem depletion method is equivalent to commercial kits which are based on immune-affinity chromatography. Moreover, this method using protein A immunoglobulin depletion was shown to be highly reproducible and a minimal amount of non-target proteins was depleted. The combination of ammonium sulfate precipitation and Protein A affinity chromatography offers a low cost, efficient, straightforward and reproducible alternative to commercial kits, with proteins remaining in native conformation, allowing protein activity and protein interaction studies.