Neuroproteomics — LC-MS Quantitative Approaches

Neuroproteomics is a scientific field that aims to study all the proteins of the central nervous system, their expression, function, and interactions. The central nervous system is intricate and heterogeneous, and the study of its proteome is consequently complex, with many biological questions still requiring deep investigation. For this, mass spectrometry approaches, most often coupled with liquid chromatography (LC-MS), have been the number one choice in proteomics, and over the years it has added many important findings to the field. At this point it is important that proteomics turns to the quantitative expression of proteins instead of only identifying which proteins are present in a given sample, much because the most important alterations may be slight alterations in the quantity of a protein in a given situation. Therefore, many LC-MS quantitative approaches have been developed relying on the labeling of the proteins or even by using label-free techniques

Impact of mesenchymal stem cells' secretome on glioblastoma pathophysiology

Background: Glioblastoma (GBM) is a highly aggressive primary brain cancer, for which curative therapies are not available. An emerging therapeutic approach suggested to have potential to target malignant gliomas has been based on the use of multipotent mesenchymal stem cells (MSCs), either unmodified or engineered to deliver anticancer therapeutic agents, as these cells present an intrinsic capacity to migrate towards malignant tumors. Nevertheless, it is still controversial whether this innate tropism of MSCs towards the tumor area is associated with cancer promotion or suppression. Considering that one of the major mechanisms by which MSCs interact with and modulate tumor cells is via secreted factors, we studied how the secretome of MSCs modulates critical hallmark features of GBM cells. Methods: The effect of conditioned media (CM) from human umbilical cord perivascular cells (HUCPVCs, a MSC population present in the Wharton's jelly of the umbilical cord) on GBM cell viability, migration, proliferation and sensitivity to temozolomide treatment of U251 and SNB-19 GBM cells was evaluated. The in vivo chicken chorioallantoic membrane (CAM) assay was used to evaluate the effect of HUCPVCs CM on tumor growth and angiogenesis. The secretome of HUCPVCs was characterized by proteomic analyses. Results: We found that both tested GBM cell lines exposed to HUCPVCs CM presented significantly higher cellular viability, proliferation and migration. In contrast, resistance of GBM cells to temozolomide chemotherapy was not significantly affected by HUCPVCs CM. In the in vivo CAM assay, CM from HUCPVCs promoted U251 and SNB-19 tumor cells growth. Proteomic analysis to characterize the secretome of HUCPVCs identified several proteins involved in promotion of cell survival, proliferation and migration, revealing novel putative molecular mediators for the effects observed in GBM cells exposed to HUCPVCs CM. Conclusions: These findings provide novel insights to better understand the interplay between GBM cells and MSCs, raising awareness to potential safety issues regarding the use of MSCs as stem-cell based therapies for GBM.The authors would like to acknowledge the funding agencies that supported this work: Fundacao para a Ciencia e Tecnologia (FCT), Portugal, projects reference: PTDC/SAU-GMG/113795/2009 (BMC); SFRH/BD/88121/2012 (JVdC); SFRH/BD/103075/2014 (EDG); IF/00601/2012 (BMC); IF/00111/2013 (AJS); SFRH/BD/81495/2011 (SIA); PTDC/NEU-NMC/0205/2012, PTDC/NEUSCC/ 7051/2014, PEst-C/SAU/LA0001/2013-2014 and UID/NEU/04539/2013 (BM); Fundacao Calouste Gulbenkian (BMC); Liga Portuguesa Contra o Cancro (BMC); " COMPETE Programa Operacional Factores de Competitividade, QREN, the European Union (FEDER-Fundo Europeu de Desenvolvimento Regional) and by The National Mass Spectrometry Network (RNEM) under the contract REDE/1506/REM/2005; FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER-007038; and project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). The funding body did not have a role in the design of the study, in collection, analysis or interpretation of data, or in writing the manuscript

Cofilin-1 is a mechanosensitive regulator of transcription

The mechanical properties of the extracellular environment are interrogated by cells and integrated through mechanotransduction. Many cellular processes depend on actomyosin-dependent contractility, which is influenced by the microenvironment's stiffness. Here, we explored the influence of substrate stiffness on the proteome of proliferating undifferentiated human umbilical cord-matrix mesenchymal stem/stromal cells. The relative abundance of several proteins changed significantly by expanding cells on soft (∼3 kPa) or stiff substrates (GPa). Many such proteins are associated with the regulation of the actin cytoskeleton, a major player of mechanotransduction and cell physiology in response to mechanical cues. Specifically, Cofilin-1 levels were elevated in cells cultured on soft comparing with stiff substrates. Furthermore, Cofilin-1 was de-phosphorylated (active) and present in the nuclei of cells kept on soft substrates, in contrast with phosphorylated (inactive) and widespread distribution in cells on stiff. Soft substrates promoted Cofilin-1-dependent increased RNA transcription and faster RNA polymerase II-mediated transcription elongation. Cofilin-1 is part of a novel mechanism linking mechanotransduction and transcription.publishe

Unveiling the Differences of Secretome of Human Bone Marrow Mesenchymal Stem Cells, Adipose Tissue-Derived Stem Cells, and Human Umbilical Cord Perivascular Cells: A Proteomic Analysis

The use of human mesenchymal stem cells (hMSCs) has emerged as a possible therapeutic strategy for CNS-related conditions. Research in the last decade strongly suggests that MSC-mediated benefits are closely related with their secretome. Studies published in recent years have shown that the secretome of hMSCs isolated from different tissue sources may present significant variation. With this in mind, the present work performed a comparative proteomic-based analysis through mass spectrometry on the secretome of hMSCs derived from bone marrow (BMSCs), adipose tissue (ASCs), and human umbilical cord perivascular cells (HUCPVCs). The results revealed that BMSCs, ASCs, and HUCPVCs differed in their secretion of neurotrophic, neurogenic, axon guidance, axon growth, and neurodifferentiative proteins, as well as proteins with neuroprotective actions against oxidative stress, apoptosis, and excitotoxicity, which have been shown to be involved in several CNS disorder/injury processes. Although important changes were observed within the secretome of the cell populations that were analyzed, all cell populations shared the capability of secreting important neuroregulatory molecules. The difference in their secretion pattern may indicate that their secretome is specific to a condition of the CNS. Nevertheless, the confirmation that the secretome of MSCs isolated from different tissue sources is rich in neuroregulatory molecules represents an important asset not only for the development of future neuroregenerative strategies but also for their use as a therapeutic option for human clinical trials.Foundation Calouste de Gulbenkian for the funds attributed to A.J.S.; Portuguese Foundation for Science and Technology (FCT) PhD fel- lowships attributed to A.O.P. (SFRH/BD/33900/2009) and S.I.A. (SFRH/BD/81495/2011) and Ciência 2007, IF Development Grant attributed to A.J.S., and projects PTDC/ NEU-NMC/0205/2012, UID/NEU/04539/2013; cofinanced by COMPETE Programa Operacional Factores de Compe- titividade; and by The National Mass Spectrometry Network (RNEM) (REDE/1506/REM/2005); Prémios Santa Casa Neurociências—Prize Melo e Castro for Spinal Cord Injury Research; cofunded by Programa Operacional Regional do Norte (ON.2–O Novo Norte),ao abrigo do Quadro de Referência Estratégico Nacional (QREN), and através do Fundo Europeu de Desenvolvimento Regional (FEDER). The authors also would like to thank Professor J.E.D. (University of Toronto, Canada) and Professor J.M.G. (Tulane University) for kindly providing HUCPVCs and ASCs, respectivelyinfo:eu-repo/semantics/publishedVersio

Crosstalk between glial and glioblastoma cells triggers the "go-or-grow" phenotype of tumor cells

Background: Glioblastoma (GBM), the most malignant primary brain tumor, leads to poor and unpredictable clinical outcomes. Recent studies showed the tumor microenvironment has a critical role in regulating tumor growth by establishing a complex network of interactions with tumor cells. In this context, we investigated how GBM cells modulate resident glial cells, particularly their paracrine activity, and how this modulation can influence back on the malignant phenotype of GBM cells. Methods: Conditioned media (CM) of primary mouse glial cultures unexposed (unprimed) or exposed (primed) to the secretome of GL261 GBM cells were analyzed by proteomic analysis. Additionally, these CM were used in GBM cells to evaluate their impact in glioma cell viability, migration capacity and activation of tumor-related intracellular pathways. Results: The proteomic analysis revealed that the pre-exposure of glial cells to CM from GBM cells led to the upregulation of several proteins related to inflammatory response, cell adhesion and extracellular structure organization within the secretome of primed glial cells. At the functional levels, CM derived from unprimed glial cells favored an increase in GBM cell migration capacity, while CM from primed glial cells promoted cells viability. These effects on GBM cells were accompanied by activation of particular intracellular cancer-related pathways, mainly the MAPK/ERK pathway, which is a known regulator of cell proliferation. Conclusions: Together, our results suggest that glial cells can impact on the pathophysiology of GBM tumors, and that the secretome of GBM cells is able to modulate the secretome of neighboring glial cells, in a way that regulates the "go-or-grow" phenotypic switch of GBM cells.Fundação para a Ciência e Tecnologia (IF/00601/2012 to B.M.C.; IF/00111 to A.J.S; SFRH/BD/52287/2013 to A.I.O.; SFRH/BD/81495/2011 to S.I.A.; SFRH/BD/88121/2012 to J.V.C.; projects PTDC/SAU-GMG/113795/2009 to B.M.C.; PTDC/NEU-NMC/0205/2012, PTDC/NEU-SCC/7051/2014, PEst-C/SAU/LA0001/2013–2014 and UID/NEU/04539/2013 to B.M.), Liga Portuguesa Contra o Cancro (B.M.C.), Fundação Calouste Gulbenkian (B.M.C.) and Inter-University Doctoral Programme in Ageing and Chronic Disease (PhDOC; to A.I.O.). Project co-financed by Programa Operacional Regional do Norte (ON.2—O Novo Norte), Quadro de Referência Estratégico Nacional (QREN), Fundo Europeu de Desenvolvimento Regional (FEDER), Programa Operacional Factores de Competitividade (COMPETE), and by The National Mass Spectrometry Network (RNEM) under the contract REDE/1506/REM/2005info:eu-repo/semantics/publishedVersio

DJ‐1 neuronal rescue under oxidative stress: implications for Parkinson's disease

Tese de doutoramento em Biociências, na especialidade de Biologia Celular e Molecular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de CoimbraMutações em vários genes, incluindo o gene DJ-1, têm sido referidas como causadores de formas hereditárias da Doença de Parkinson (DP), sendo que a partir do estudo das proteínas codificadas por esses genes já foram obtidas informações relevantes sobre os mecanismos moleculares da degeneração dopaminérgica que caracteriza esta patologia. De entre esses mecanismos o stress oxidativo tem-se evidenciado, sendo apontado como a principal causa da morte dos neurónios dopaminérgicos independentemente dos estímulos iniciais que levam à doença. A proteína DJ-1, associada a uma das formas hereditárias da DP, tem sido considerada como uma proteína de resposta ao stress oxidativo que apresenta um papel importante na protecção contra este estímulo. Para além disso, diversas funções têm-lhe sido propostas, as quais têm contribuído para aumentar o conhecimento sobre os mecanismos envolvidos na neurodegeneração associada a stress oxidativo, observada em doentes com DP. A proteína DJ-1 é considerada uma proteína multifuncional, contudo os mecanismos pelos quais esta exerce a sua função, assim como os mecanismos responsáveis pela sua regulação, não estão totalmente compreendidos. Assim, será espectável que ao se elucidar a função fisiológica da DJ-1 sejam também alcançados importantes conhecimentos sobre a DP. Além disso, o stress oxidativo é crucial para a actividade da DJ-1, encontrando-se associado à regulação da maioria dos mecanismos de acção e das interacções estabelecidas por esta. Contudo, os mechanismos exactos responsáveis pela regulação da proteína DJ-1 pelo stress oxidativo, assim como muitos dos seus interactores continuam por identificar. Nesse sentido, com este projecto pretendeu-se elucidar os mecanismos de acção da DJ-1. Mais precisamente pretendeu clarificar os mecanismos pelos quais esta protéina exerce a sua capacidade neuroprotectora a curto e longo prazo, através da identificação dos seus interactores e da dinâmica das interacções durante o stress oxidativo. Para alcançar os objectivos pretendidos, foi realizado um estudo exaustivo de interactómica que permitiu identificar e quantificar 881 proteínas envolvidas na rede de interacções estabelecidas por intermédio da actividade da DJ-1 em condições de stress oxidativo. Este estudo foi realizado em condições bem definidas associadas à activação de duas importantes vias de sobrevivência centrais para a capacidade neuroprotectora da DJ-1 (as vias da ERK1/2 e PI3-K/Akt), e com importantes implicações para o estudo da DJ-1 no contexto da DP dado que foi observado um impacto evidente na actividade mitocondrial causado pelas condições experimentais usadas. Com vista a uma melhor caracterização da natureza dinâmica das interacções estabelecidas foi desenvolvida uma estratégia baseada na optimização da análise SWATH-MS que consistiu no uso de um método de digestão em gel com elevada reprodutibilidade e eficiência, designado Short-GeLC, combinado com o uso de proteínas recombinantes como padrão interno. Esta análise permitiu traçar perfis de interacção revelando grupos de proteínas com o mesmo tipo de modulação que também partilhavam actividades biológicas similares, apontado para a implicação da DJ-1 na modulação desses mecanismos e contribuindo assim para clarificar a sua actividade neuroprotectora. A rede de interacções identificada neste trabalho contribuiu em grande parte para aumentar o conhecimento associado às vias envolvidas na resposta da DJ-1 ao stress oxidativo, que podem ser divididas de um modo geral em dois tipos de resposta: um correspondendo a uma resposta mais imediata que está principalmente associada à identificação de proteínas de resposta ao stress tais como oxireductases e fosfatases; e outro mecanismo associado a uma resposta adaptativa e de longo prazo que está associada com a forte representação de proteínas envolvidas em expressão de genes. Para além disso, foi possível confirmar que: i) a proteína DJ-1 para além de actuar em diferentes vias, também controla o mesmo mecanismo a diversos níveis, tal como no caso da regulação da expressão de proteínas e das vias intrínsecas e extrínsecas de apoptose, e ii) apontar pela primeira vez para o envolvimento da DJ-1 com grânulos de stress e na resposta a danos no ADN, cuja importância para a viabilidade celular e implicação nos mecanismos de neurodegeneração, incluindo os envolvidos em DP, tem vindo a aumentar entre a comunidade científica. Em conclusão, este trabalho resultou na identificação de várias novas proteínas envolvidas na rede de interacções da DJ-1 contribuíndo para a elucidação dos mecanismos de protecção neuronal mediados pela DJ-1 contra o stress oxidativo, e apontando novos mecanismos. Para além disso, muitas da proteínas identificadas estão bem estabelecidas em várias funções celulares implicadas na DP. Deste modo, estes resultados contribuem também para um melhor entendimento da DP e dos vários mecanismos envolvidos no estabelecimento e progressão da doença. Mutations in several genes, including DJ‐1 gene, have been reported to cause hereditary forms of Parkinson’s Disease (PD), and in fact the study of these genes has already provided valuable insights into the molecular mechanisms of dopaminergic degeneration characteristic of this pathology, from which oxidative stress has been considered the cause of dopaminergic neurons death in PD independently of the trigger of the disease. The PD‐ associated protein DJ‐1 has been recognized as a redox response protein with an important role in the protection against the oxidative stress insults. Moreover, several functions have been reported for DJ‐1 which have contributed with significant insights into the mechanisms of oxidative stress‐related neurodegeneration observed in PD patients. Considering all these evidences, DJ‐1 was considered as a multifunctional protein although its exact mechanism of action and regulation remains largely unknown. It is therefore expected that by elucidating DJ‐1 physiological function important insights into PD will also be achieved. Moreover, oxidative stress conditions seem to be crucial for DJ‐1 activity, being associated with the regulation of the majority of its mechanisms of action and the interactions established by it. However, all the exact mechanisms behind oxidative stress regulation of DJ‐1 and many of the interaction partners of DJ‐1 remain unidentified. Therefore, this project proposed to elucidate the mechanisms of action of DJ‐1. More specifically, aimed to clarify the mechanisms by which DJ‐1 modulate short‐term and long‐term neuroprotection, by identifying the binding partners of DJ‐1 during oxidative stress and the dynamics of these interactions under oxidative stress. To achieve these goals, a comprehensive interactomic study of DJ‐1 under oxidative stress conditions was conducted, allowing the identification and quantification of 881 proteins involved in the network of interactions established via DJ‐1 activity. Moreover, this study was performed in a well‐defined model associated with the activation of two key survival pathways central for DJ‐1 neuroprotective function (ERK1/2 and PI3‐K/Akt pathways), and with important implications for the study of DJ‐1 role in Parkinson’s Disease context, since an evident impact in the mitochondrial activity was also reflected in the experimental conditions used. More importantly, the optimized SWATH‐MS based pipeline developed consisting in the use of a high reproducible and efficient in‐gel digestion methods, designed as short‐GeLC, combined with the use of recombinant proteins as internal standard, was applied to characterize the dynamic nature of the interactions. The SWATH‐MS analysis allowed to trace the profiles of interaction revealing clusters of proteins with the same type of modulation that also shared similar biological activities, pointing out for an implication of DJ‐1 in those mechanisms and largely contributing to clarify the DJ‐1 neuroprotective activity. In fact, the network of interactors identified in this work largely contributes to extend the knowledge regarding the pathways involved in DJ‐1 response to oxidative stress, which can be generically divided in two different types: one corresponding with a more immediate response, associated with the identification of stress responsive proteins such as oxireductases and phosphatases; and the other corresponding to an adaptive and long term response associated with the strong representation of proteins involved in gene expression. Moreover, it was possible to confirmed that: i) DJ‐1 not only acts at different pathways but also controls the same mechanisms at different stages, such as in the case of the regulation of protein expression and of the intrinsic and extrinsic apoptotic pathways, and ii) to point out for the first time, the involvement of DJ‐1 with stress granules and with the response to DNA damage, which relevance for cell survival and implication in neurodegeneration (including in PD) is increasing in importance within the scientific community. In conclusion, this study resulted in the identification of several novel proteins involved in DJ‐1 network contributing to the elucidation of DJ‐1‐mediated neuronal protection against oxidative stress, and pointing out some new mechanisms. In addition, many of the proteins identified are well established in distinct cellular functions implicated in PD. Thus, these results may also contribute to a better understanding of PD and the distinct pathways involved in the establishment and progression of the disease.Mutations in several genes, including DJ‐1 gene, have been reported to cause hereditary forms of Parkinson’s Disease (PD), and in fact the study of these genes has already provided valuable insights into the molecular mechanisms of dopaminergic degeneration characteristic of this pathology, from which oxidative stress has been considered the cause of dopaminergic neurons death in PD independently of the trigger of the disease. The PD‐ associated protein DJ‐1 has been recognized as a redox response protein with an important role in the protection against the oxidative stress insults. Moreover, several functions have been reported for DJ‐1 which have contributed with significant insights into the mechanisms of oxidative stress‐related neurodegeneration observed in PD patients. Considering all these evidences, DJ‐1 was considered as a multifunctional protein although its exact mechanism of action and regulation remains largely unknown. It is therefore expected that by elucidating DJ‐1 physiological function important insights into PD will also be achieved. Moreover, oxidative stress conditions seem to be crucial for DJ‐1 activity, being associated with the regulation of the majority of its mechanisms of action and the interactions established by it. However, all the exact mechanisms behind oxidative stress regulation of DJ‐1 and many of the interaction partners of DJ‐1 remain unidentified. Therefore, this project proposed to elucidate the mechanisms of action of DJ‐1. More specifically, aimed to clarify the mechanisms by which DJ‐1 modulate short‐term and long‐term neuroprotection, by identifying the binding partners of DJ‐1 during oxidative stress and the dynamics of these interactions under oxidative stress. To achieve these goals, a comprehensive interactomic study of DJ‐1 under oxidative stress conditions was conducted, allowing the identification and quantification of 881 proteins involved in the network of interactions established via DJ‐1 activity. Moreover, this study was performed in a well‐defined model associated with the activation of two key survival pathways central for DJ‐1 neuroprotective function (ERK1/2 and PI3‐K/Akt pathways), and with important implications for the study of DJ‐1 role in Parkinson’s Disease context, since an evident impact in the mitochondrial activity was also reflected in the experimental conditions used. More importantly, the optimized SWATH‐MS based pipeline developed consisting in the use of a high reproducible and efficient in‐gel digestion methods, designed as short‐GeLC, combined with the use of recombinant proteins as internal standard, was applied to characterize the dynamic nature of the interactions. The SWATH‐MS analysis allowed to trace the profiles of interaction revealing clusters of proteins with the same type of modulation that also shared similar biological activities, pointing out for an implication of DJ‐1 in those mechanisms and largely contributing to clarify the DJ‐1 neuroprotective activity. In fact, the network of interactors identified in this work largely contributes to extend the knowledge regarding the pathways involved in DJ‐1 response to oxidative stress, which can be generically divided in two different types: one corresponding with a more immediate response, associated with the identification of stress responsive proteins such as oxireductases and phosphatases; and the other corresponding to an adaptive and long term response associated with the strong representation of proteins involved in gene expression. Moreover, it was possible to confirmed that: i) DJ‐1 not only acts at different pathways but also controls the same mechanisms at different stages, such as in the case of the regulation of protein expression and of the intrinsic and extrinsic apoptotic pathways, and ii) to point out for the first time, the involvement of DJ‐1 with stress granules and with the response to DNA damage, which relevance for cell survival and implication in neurodegeneration (including in PD) is increasing in importance within the scientific community. In conclusion, this study resulted in the identification of several novel proteins involved in DJ‐1 network contributing to the elucidation of DJ‐1‐mediated neuronal protection against oxidative stress, and pointing out some new mechanisms. In addition, many of the proteins identified are well established in distinct cellular functions implicated in PD. Thus, these results may also contribute to a better understanding of PD and the distinct pathways involved in the establishment and progression of the disease.Rede Nacional de Espectrometria de Massa (RNEM) - REDE/1506/REM/200

Modulation of signaling pathways by DJ-1: An updated overview

Efforts have been made to understand the physiological and pathological role of DJ-1, a Parkinson's disease (PD)-associated protein, to provide new insights into PD pathophysiology. Such studies have revealed several neuroprotective roles of DJ-1, from which its ability to modulate signaling pathways seems to be of utmost importance for cell death regulation by DJ-1. Indeed, research on these topics has led to a higher number of publications disclosing a variety of mechanisms through which DJ-1 is able to modulate signaling pathways in distinct disease-related contexts. Thus, this graphical review presents the most relevant findings concerning the mechanisms through which DJ-1 exerts its regulatory activity on signaling cascades relevant for DJ-1 neuroprotective action, namely ERK1/2, PI3K/Akt, and ASK1 pathways, and Nrf2 and p53 transcription factors-related signaling. A greater focus was given to perform an overview of the research interests over the last years, especially in the most recent works, to highlight the current research lines in this topic, and point out future directions in the field. In addition, the impact of DJ-1 mutations causative of PD and the importance of the redox status of DJ-1's cysteine residues for the action of DJ-1 on signaling modulation was also addressed to uncover the potential pathological mechanisms associated with loss of DJ-1 native function

Bursaphelenchus xylophilus and B. mucronatus secretomes: a comparative proteomic analysis

The pinewood nematode, Bursaphelenchus xylophilus, recognized as a worldwide major forest pest, is a migratory endoparasitic nematode with capacity to feed on pine tissues and also on fungi colonizing the trees. Bursaphelenchus mucronatus, the closest related species, differs from B. xylophilus on its pathogenicity, making this nematode a good candidate for comparative analyses. Secretome profiles of B. xylophilus and B. mucronatus were obtained and proteomic differences were evaluated by quantitative SWATH-MS. From the 681 proteins initially identified, 422 were quantified and compared between B. xylophilus and B. mucronatus secretomes and from these, 243 proteins were found differentially regulated: 158 and 85 proteins were increased in B. xylophilus and B. mucronatus secretomes, respectively. While increased proteins in B. xylophilus secretome revealed a strong enrichment in proteins with peptidase activity, the increased proteins in B. mucronatus secretome were mainly related to oxidative stress responses. The changes in peptidases were evaluated at the transcription level by RT-qPCR, revealing a correlation between the mRNA levels of four cysteine peptidases with secretion levels. The analysis presented expands our knowledge about molecular basis of B. xylophilus and B. mucronatus hosts interaction and supports the hypothesis of a key role of secreted peptidases in B. xylophilus pathogenicity

Comparative Analysis of Bursaphelenchus xylophilus Secretome Under Pinus pinaster and P. pinea Stimuli

The pinewood nematode (PWN), Bursaphelenchus xylophilus, the pine wilt disease's (PWD) causal agent, is a migratory endoparasitic nematode skilled to feed on pine tissues and on fungi that colonize the trees. In order to study B. xylophilus secretomes under the stimulus of pine species with different susceptibilities to disease, nematodes were exposed to aqueous pine extracts from Pinus pinaster (high-susceptible host) and P. pinea (low-susceptible host). Sequential windowed acquisition of all theoretical mass spectra (SWATH-MS) was used to determine relative changes in protein amounts between B. xylophilus secretions, and a total of 776 secreted proteins were quantified in both secretomes. From these, 22 proteins were found increased in the B. xylophilus secretome under the P. pinaster stimulus and 501 proteins increased under the P. pinea stimulus. Functional analyses of the 22 proteins found increased in the P. pinaster stimulus showed that proteins with peptidase, hydrolase, and antioxidant activities were the most represented. On the other hand, gene ontology (GO) enrichment analysis of the 501 proteins increased under the P. pinea stimulus revealed an enrichment of proteins with binding activity. The differences detected in the secretomes highlighted the diverse responses from the nematode to overcome host defenses with different susceptibilities and provide new clues on the mechanism behind the pathogenicity of this plant-parasitic nematode. Proteomic data are available via ProteomeXchange with identifier PXD024011

Proteomic analysis of tissue-specific protein aggregation signatures throughout mammalian aging

Proteome and proteostasis network ( disruptions lead to proteostasis imbalances and accumulation of protein aggregates, characteristic of several age related diseases 1 3 The consequences of PN disturbances in the context of healthy aging remains to be thoroughly studied in mammals To determine whether accumulation of insoluble proteins occurs during mammalian aging, young adult 6 month old), middle aged 13 month old), and old 18 and 24 month old) C 57 BL/ 6 mice were used to obtain detergent insoluble fractions Detergent insoluble and soluble fractions were isolated from total protein extracts of the liver, followed by characterization of total, detergent soluble, and detergent insoluble protein profiles We performed SWATH mass spectrometry analysis to identify differential proteome signatures of aging and determine which proteins are more prone to aggregate and further elucidated the altered functions and biological processes.publishe