Enrico Massignani - Academia.edu (original) (raw)
Papers by Enrico Massignani
Methods in molecular biology, Nov 13, 2022
Journal of Visualized Experiments
Experimental and Molecular Therapeutics, 2019
Mutations in RNA splicing factors commonly occur in myeloid leukemia and solid tumors. These muta... more Mutations in RNA splicing factors commonly occur in myeloid leukemia and solid tumors. These mutations occur in a heterozygous manner and confer dependency on the wild-type allele. Studies have shown that splicing factor mutant cancers are vulnerable to further perturbation of splicing, by pharmacological intervention that directly targets core splicing factors. Our project identifies the use of PRMT inhibitors, as plausible alternative therapeutic strategy to treat spliceosomal mutant leukemia. The data show that splicing factor mutant leukemia exhibit greater sensitivity to the use of inhibitors against Type I PRMTs and PRMT5, in comparison to their wild-type counterparts. As the need for new therapeutic strategies in cancer treatment increases, this study identifies PRMT inhibitors as a potential therapeutic intervention against cancers with splicing factor mutations. Citation Format: Jia Yi Fong, Diana Low, Luca Pignata, Kimihito Cojin Kawabata, Stanley CW Lee, Cheryl Koh, Daniele Musiani, Enrico Massignani, Cheng Mun Wun, Pierre-Alexis V. Goy, Yudao Shen, Heike Wollmann, Florence PH Gay, Genna Luciani, Dalia Barsyte, Jian Jin, Ari M. Melnick, Tiziana Bonaldi, Omar Abdel-Wahab, Ernesto Guccione. Therapeutic targeting of RNA splicing through inhibition of protein arginine methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4731.
A post-translational modifications (PTM) is the covalent modification of a protein after its synt... more A post-translational modifications (PTM) is the covalent modification of a protein after its synthesis, by either addition or removal of functional groups. PTMs significantly increase the complexity of the proteome by allowing each protein to exist in different forms, which can have different activity, stability or binding specificity. Because of their central role in protein regulation, PTMs are often deregulated in many diseases, especially cancer. Since all PTMs introduce a change in the proteins mass, one of the leading techniques to study PTMs is Tandem Mass Spectrometry (MS/MS), which can measure the mass of molecules with high precision and resolution. Here, we apply computational methods to MS data, in order to study PTMs from two points of view. In one project, we conducted a comprehensive analysis of Arginine (R) methylation at a global level. To achieve this, we significantly improved hmSEEKER, our in-house developed computational tool for the analysis of MS data from hea...
Nucleic Acids Research
MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by... more MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by the activity of the Microprocessor and its associated proteins. Through high resolution mass spectrometry (MS)- proteomics we discovered that this complex is extensively methylated, with 84 methylated sites associated to 19 out of its 24 subunits. The majority of the modifications occurs on arginine (R) residues (61), leading to 81 methylation events, while 30 lysine (K)-methylation events occurs on 23 sites of the complex. Interestingly, both depletion and pharmacological inhibition of the Type-I Protein Arginine Methyltransferases (PRMTs) lead to a widespread change in the methylation state of the complex and induce global decrease of miRNA expression, as a consequence of the impairment of the pri-to-pre-miRNA processing step. In particular, we show that the reduced methylation of the Microprocessor subunit ILF3 is linked to its diminished binding to the pri-miRNAs miR-15a/16, miR-17–...
Current protein & peptide science, 2020
The absence of efficient mass spectrometry-based approaches for the large-scale analysis of prote... more The absence of efficient mass spectrometry-based approaches for the large-scale analysis of protein arginine methylation has hindered the understanding of its biological role, beyond the transcriptional regulation occurring through histone modification. In the last decade, however, several technological advances of both the biochemical methods for methylated polypeptide enrichment and the computational pipelines for MS data analysis have considerably boosted this research field, generating novel insights about the extent and role of this post translational modification. Here, we offer an overview on state-of-the-art approaches for the high-confidence identification and accurate quantification of protein arginine methylation by high-resolution mass spectrometry methods, which comprise the development of both biochemical and bioinformatics methods. The further optimization and systematic application of these analytical solutions will lead to groundbreaking discoveries on the role of p...
Frontiers in Molecular Biosciences, 2021
RNA binding proteins (RBPs) bind RNAs through specific RNA-binding domains, generating multi-mole... more RNA binding proteins (RBPs) bind RNAs through specific RNA-binding domains, generating multi-molecular complexes known as ribonucleoproteins (RNPs). Various post-translational modifications (PTMs) have been described to regulate RBP structure, subcellular localization, and interactions with other proteins or RNAs. Recent proteome-wide experiments showed that RBPs are the most representative group within the class of arginine (R)-methylated proteins. Moreover, emerging evidence suggests that this modification plays a role in the regulation of RBP-RNA interactions. Nevertheless, a systematic analysis of how changes in protein-R-methylation can affect globally RBPs-RNA interactions is still missing. We describe here a quantitative proteomics approach to profile global changes of RBP-RNA interactions upon the modulation of type I and II protein arginine methyltransferases (PRMTs). By coupling the recently described Orthogonal Organic Phase Separation (OOPS) strategy with the Stable Isot...
bioRxiv, 2021
The histone de-methylase LSD1 is over-expressed in haematological tumours and has emerged as a pr... more The histone de-methylase LSD1 is over-expressed in haematological tumours and has emerged as a promising target for anti-cancer treatment, so that several LSD1 inhibitors are under development and testing, in pre-clinical and clinical settings. However, the complete understanding of their complex mechanism of action is still unreached. Here, we unravelled a novel mode of action of the LSD1 inhibitors MC2580 and DDP-38003, showing that they can induce differentiation of AML cells through the down-regulation of the chromatin protein GSE1. Analysis of the phenotypic effects of GSE1 depletion in NB4 cells showed a strong decrease of cell viability in vitro and of tumour growth in vivo. Mechanistically, we found that a set of genes associated with immune response and cytokine signalling pathways are up-regulated by LSD1 inhibitors through GSE1 protein reduction and that LSD1 and GSE1 co-localise at promoters of a subset of these genes at the basal state, enforcing their transcriptional s...
Cell Reports, 2020
Highlights d Cisplatin leads to increased PRMT1 association to chromatin and H4R3 methylation d P... more Highlights d Cisplatin leads to increased PRMT1 association to chromatin and H4R3 methylation d PRMT1 increase in chromatin is mediated by DNA-PK d Chromatin-associated PRMT1 sustains the transcription of SASP genes d Inhibition or genetic depletion of PRMT1 blocks SASP and sensitizes cancer cells to cisplatin
Science Signaling
Protein arginine methyltransferases (PRMTs) catalyze arginine methylation on both chromatin-bound... more Protein arginine methyltransferases (PRMTs) catalyze arginine methylation on both chromatin-bound and cytoplasmic proteins. Accumulating evidence supports the involvement of PRMT5, the major type II PRMT, in cell survival and differentiation pathways that are important during development and in tumorigenesis. PRMT5 is an attractive drug target in various cancers, and inhibitors are currently in oncological clinical trials. Nonetheless, given the complex biology of PRMT5 and its multiple nonhistone substrates, it is paramount to fully characterize these dynamic changes in methylation and to link them to the observed anticancer effects to fully understand the functions of PRMT5 and the consequences of its inhibition. Here, we used a newly established pipeline coupling stable isotope labeling with amino acids in cell culture (SILAC) with immunoenriched methyl peptides to globally profile arginine monomethylation and symmetric dimethylation after PRMT5 inhibition by a selective inhibito...
MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by... more MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by the activity of the Large Drosha Complex (LDC). Through high resolution mass spectrometry (MS) analysis we discovered that the LDC is extensively methylated, with 82 distinct methylated sites associated to 16 out of 23 subunits of the LDC. The majority of these modifications occurs on arginine (R)- residues (61), leading to 86 methylation events, while 29 lysine (K)-methylation events occurs on 21 sites of the complex. Interestingly, the depletion and pharmacological inhibition of PRMT1 lead to a widespread alteration of the methylation state of the complex and induce global decrease of miRNA expression, as a consequence of the specific impairment of the pri-to-pre-miRNA processing step. In particular, we show that the reduced methylation of the ILF3 subunit of the complex is linked to its diminished binding to the target pri-miRNAs. Overall, our study uncovers a previously uncharacteri...
Protein Arginine (R) methylation is a post-translational modification involved in various biologi... more Protein Arginine (R) methylation is a post-translational modification involved in various biological processes, such as RNA splicing, DNA repair, immune response, signal transduction, and tumour development. Although several advancements were made in the study of this modification by mass spectrometry, researchers still face the problem of a high false discovery rate. We present a dataset of high-quality methylations obtained from several different heavy methyl SILAC (hmSILAC) experiments analysed with a machine learning-based tool doublets and show that this model allows for improved high-confidence identification of real methyl-peptides. Overall, our results are consistent with the notion that protein R methylation modulates protein:RNA interactions and suggest a role in rewiring protein:protein interactions, for which we provide experimental evidence for a representative case (i.e. NONO:PSPC1). Upon intersecting our R-methyl-sites dataset with a phosphosites dataset, we observed ...
PROTEOMICS
Heavy methyl Stable Isotope Labeling with Amino acids in Cell culture (hmSILAC) is a metabolic la... more Heavy methyl Stable Isotope Labeling with Amino acids in Cell culture (hmSILAC) is a metabolic labeling strategy employed in proteomics to increase the confidence of global identification of methylated peptides by MS. However, to this day, the automatic and robust identification of heavy and light peak doublets from MS-raw data of hmSILAC experiments is a challenging task, for which the choice of computational methods is very limited. Here, hmSEEKER, a software designed to work downstream of a MaxQuant analysis for in-depth search of MS peak pairs that correspond to light and heavy methyl-peptide within MaxQuant-generated tables is described with good sensitivity and specificity. The software is written in Perl, and its code and user manual are freely available at Bitbucket (https://bit.ly/2scCT9u). Protein methylation is a posttranslational modification (PTM) consisting in the addition of one or more methyl-groups to arginine and lysine residues of a protein. Lysine can be mono-, di-, or tri-methylated, while arginine can be mono-or di-methylated. Arginine di-methylation can, in turn, be either symmetrical or asymmetrical. While the functional implication of histone lysine and arginine methylation in the regulation of gene expression is well established, recent evidence revealed that this PTM is widespread at the proteome level, a notion that expands its regulatory potential well beyond chromatin structure and
Methods in molecular biology, Nov 13, 2022
Journal of Visualized Experiments
Experimental and Molecular Therapeutics, 2019
Mutations in RNA splicing factors commonly occur in myeloid leukemia and solid tumors. These muta... more Mutations in RNA splicing factors commonly occur in myeloid leukemia and solid tumors. These mutations occur in a heterozygous manner and confer dependency on the wild-type allele. Studies have shown that splicing factor mutant cancers are vulnerable to further perturbation of splicing, by pharmacological intervention that directly targets core splicing factors. Our project identifies the use of PRMT inhibitors, as plausible alternative therapeutic strategy to treat spliceosomal mutant leukemia. The data show that splicing factor mutant leukemia exhibit greater sensitivity to the use of inhibitors against Type I PRMTs and PRMT5, in comparison to their wild-type counterparts. As the need for new therapeutic strategies in cancer treatment increases, this study identifies PRMT inhibitors as a potential therapeutic intervention against cancers with splicing factor mutations. Citation Format: Jia Yi Fong, Diana Low, Luca Pignata, Kimihito Cojin Kawabata, Stanley CW Lee, Cheryl Koh, Daniele Musiani, Enrico Massignani, Cheng Mun Wun, Pierre-Alexis V. Goy, Yudao Shen, Heike Wollmann, Florence PH Gay, Genna Luciani, Dalia Barsyte, Jian Jin, Ari M. Melnick, Tiziana Bonaldi, Omar Abdel-Wahab, Ernesto Guccione. Therapeutic targeting of RNA splicing through inhibition of protein arginine methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4731.
A post-translational modifications (PTM) is the covalent modification of a protein after its synt... more A post-translational modifications (PTM) is the covalent modification of a protein after its synthesis, by either addition or removal of functional groups. PTMs significantly increase the complexity of the proteome by allowing each protein to exist in different forms, which can have different activity, stability or binding specificity. Because of their central role in protein regulation, PTMs are often deregulated in many diseases, especially cancer. Since all PTMs introduce a change in the proteins mass, one of the leading techniques to study PTMs is Tandem Mass Spectrometry (MS/MS), which can measure the mass of molecules with high precision and resolution. Here, we apply computational methods to MS data, in order to study PTMs from two points of view. In one project, we conducted a comprehensive analysis of Arginine (R) methylation at a global level. To achieve this, we significantly improved hmSEEKER, our in-house developed computational tool for the analysis of MS data from hea...
Nucleic Acids Research
MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by... more MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by the activity of the Microprocessor and its associated proteins. Through high resolution mass spectrometry (MS)- proteomics we discovered that this complex is extensively methylated, with 84 methylated sites associated to 19 out of its 24 subunits. The majority of the modifications occurs on arginine (R) residues (61), leading to 81 methylation events, while 30 lysine (K)-methylation events occurs on 23 sites of the complex. Interestingly, both depletion and pharmacological inhibition of the Type-I Protein Arginine Methyltransferases (PRMTs) lead to a widespread change in the methylation state of the complex and induce global decrease of miRNA expression, as a consequence of the impairment of the pri-to-pre-miRNA processing step. In particular, we show that the reduced methylation of the Microprocessor subunit ILF3 is linked to its diminished binding to the pri-miRNAs miR-15a/16, miR-17–...
Current protein & peptide science, 2020
The absence of efficient mass spectrometry-based approaches for the large-scale analysis of prote... more The absence of efficient mass spectrometry-based approaches for the large-scale analysis of protein arginine methylation has hindered the understanding of its biological role, beyond the transcriptional regulation occurring through histone modification. In the last decade, however, several technological advances of both the biochemical methods for methylated polypeptide enrichment and the computational pipelines for MS data analysis have considerably boosted this research field, generating novel insights about the extent and role of this post translational modification. Here, we offer an overview on state-of-the-art approaches for the high-confidence identification and accurate quantification of protein arginine methylation by high-resolution mass spectrometry methods, which comprise the development of both biochemical and bioinformatics methods. The further optimization and systematic application of these analytical solutions will lead to groundbreaking discoveries on the role of p...
Frontiers in Molecular Biosciences, 2021
RNA binding proteins (RBPs) bind RNAs through specific RNA-binding domains, generating multi-mole... more RNA binding proteins (RBPs) bind RNAs through specific RNA-binding domains, generating multi-molecular complexes known as ribonucleoproteins (RNPs). Various post-translational modifications (PTMs) have been described to regulate RBP structure, subcellular localization, and interactions with other proteins or RNAs. Recent proteome-wide experiments showed that RBPs are the most representative group within the class of arginine (R)-methylated proteins. Moreover, emerging evidence suggests that this modification plays a role in the regulation of RBP-RNA interactions. Nevertheless, a systematic analysis of how changes in protein-R-methylation can affect globally RBPs-RNA interactions is still missing. We describe here a quantitative proteomics approach to profile global changes of RBP-RNA interactions upon the modulation of type I and II protein arginine methyltransferases (PRMTs). By coupling the recently described Orthogonal Organic Phase Separation (OOPS) strategy with the Stable Isot...
bioRxiv, 2021
The histone de-methylase LSD1 is over-expressed in haematological tumours and has emerged as a pr... more The histone de-methylase LSD1 is over-expressed in haematological tumours and has emerged as a promising target for anti-cancer treatment, so that several LSD1 inhibitors are under development and testing, in pre-clinical and clinical settings. However, the complete understanding of their complex mechanism of action is still unreached. Here, we unravelled a novel mode of action of the LSD1 inhibitors MC2580 and DDP-38003, showing that they can induce differentiation of AML cells through the down-regulation of the chromatin protein GSE1. Analysis of the phenotypic effects of GSE1 depletion in NB4 cells showed a strong decrease of cell viability in vitro and of tumour growth in vivo. Mechanistically, we found that a set of genes associated with immune response and cytokine signalling pathways are up-regulated by LSD1 inhibitors through GSE1 protein reduction and that LSD1 and GSE1 co-localise at promoters of a subset of these genes at the basal state, enforcing their transcriptional s...
Cell Reports, 2020
Highlights d Cisplatin leads to increased PRMT1 association to chromatin and H4R3 methylation d P... more Highlights d Cisplatin leads to increased PRMT1 association to chromatin and H4R3 methylation d PRMT1 increase in chromatin is mediated by DNA-PK d Chromatin-associated PRMT1 sustains the transcription of SASP genes d Inhibition or genetic depletion of PRMT1 blocks SASP and sensitizes cancer cells to cisplatin
Science Signaling
Protein arginine methyltransferases (PRMTs) catalyze arginine methylation on both chromatin-bound... more Protein arginine methyltransferases (PRMTs) catalyze arginine methylation on both chromatin-bound and cytoplasmic proteins. Accumulating evidence supports the involvement of PRMT5, the major type II PRMT, in cell survival and differentiation pathways that are important during development and in tumorigenesis. PRMT5 is an attractive drug target in various cancers, and inhibitors are currently in oncological clinical trials. Nonetheless, given the complex biology of PRMT5 and its multiple nonhistone substrates, it is paramount to fully characterize these dynamic changes in methylation and to link them to the observed anticancer effects to fully understand the functions of PRMT5 and the consequences of its inhibition. Here, we used a newly established pipeline coupling stable isotope labeling with amino acids in cell culture (SILAC) with immunoenriched methyl peptides to globally profile arginine monomethylation and symmetric dimethylation after PRMT5 inhibition by a selective inhibito...
MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by... more MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by the activity of the Large Drosha Complex (LDC). Through high resolution mass spectrometry (MS) analysis we discovered that the LDC is extensively methylated, with 82 distinct methylated sites associated to 16 out of 23 subunits of the LDC. The majority of these modifications occurs on arginine (R)- residues (61), leading to 86 methylation events, while 29 lysine (K)-methylation events occurs on 21 sites of the complex. Interestingly, the depletion and pharmacological inhibition of PRMT1 lead to a widespread alteration of the methylation state of the complex and induce global decrease of miRNA expression, as a consequence of the specific impairment of the pri-to-pre-miRNA processing step. In particular, we show that the reduced methylation of the ILF3 subunit of the complex is linked to its diminished binding to the target pri-miRNAs. Overall, our study uncovers a previously uncharacteri...
Protein Arginine (R) methylation is a post-translational modification involved in various biologi... more Protein Arginine (R) methylation is a post-translational modification involved in various biological processes, such as RNA splicing, DNA repair, immune response, signal transduction, and tumour development. Although several advancements were made in the study of this modification by mass spectrometry, researchers still face the problem of a high false discovery rate. We present a dataset of high-quality methylations obtained from several different heavy methyl SILAC (hmSILAC) experiments analysed with a machine learning-based tool doublets and show that this model allows for improved high-confidence identification of real methyl-peptides. Overall, our results are consistent with the notion that protein R methylation modulates protein:RNA interactions and suggest a role in rewiring protein:protein interactions, for which we provide experimental evidence for a representative case (i.e. NONO:PSPC1). Upon intersecting our R-methyl-sites dataset with a phosphosites dataset, we observed ...
PROTEOMICS
Heavy methyl Stable Isotope Labeling with Amino acids in Cell culture (hmSILAC) is a metabolic la... more Heavy methyl Stable Isotope Labeling with Amino acids in Cell culture (hmSILAC) is a metabolic labeling strategy employed in proteomics to increase the confidence of global identification of methylated peptides by MS. However, to this day, the automatic and robust identification of heavy and light peak doublets from MS-raw data of hmSILAC experiments is a challenging task, for which the choice of computational methods is very limited. Here, hmSEEKER, a software designed to work downstream of a MaxQuant analysis for in-depth search of MS peak pairs that correspond to light and heavy methyl-peptide within MaxQuant-generated tables is described with good sensitivity and specificity. The software is written in Perl, and its code and user manual are freely available at Bitbucket (https://bit.ly/2scCT9u). Protein methylation is a posttranslational modification (PTM) consisting in the addition of one or more methyl-groups to arginine and lysine residues of a protein. Lysine can be mono-, di-, or tri-methylated, while arginine can be mono-or di-methylated. Arginine di-methylation can, in turn, be either symmetrical or asymmetrical. While the functional implication of histone lysine and arginine methylation in the regulation of gene expression is well established, recent evidence revealed that this PTM is widespread at the proteome level, a notion that expands its regulatory potential well beyond chromatin structure and