Entamoeba histolytica causes amoebiasis which is a major health concern in developing countries. E. histolytica pathogenicity has been implicated to a large repertoire of small GTPases which switch ...between the inactive GDP bound state and the active GTP bound state with the help of guanine nucleotide exchange factors (GEFs) and GTPase activating protein (GAPs). Rho family of small GTPases are well known to modulate the actin cytoskeletal dynamics which plays a major role in E. histolytica pathogenicity. Here, we report an atypical amoebic RhoGEF, and its preferred substrate EhRho6, which, upon overexpression abrogated the pathogenic behavior of the amoeba such as adhesion to host cell, monolayer destruction, erythrophagocytosis, and formation of actin dots. A causative immunoblot analysis revealed actin degradation in the EhRho6 overexpressing trophozoites that could be inhibited by blocking the amoebic proteasomal pathway. A careful analysis of the results from a previously published transcriptomics study, in conjunction with our observations, led to the identification of a clade of Rho GTPases in this pathogenic amoeba which we hypothesize to have implications during the amoebic encystation.
The transmission of Entamoeba histolytica from one host to another depends on its ability to undergo encystation. In this study, we identified a novel EhRhoGEFduo and its substrate RhoGTPases which are involved in the actin degradation and we hypothesize its implications in amoebic encystation. This study is the first report of Rho family members associated with the degradation of actin in E. histolytica.
To acquire a deeper understanding of malignant melanoma (MM), it is essential to study the proteome of patient tissues. In particular, phosphoproteomics of MM has become of significant importance ...because of the central role that phosphorylation plays in the development of MM. Investigating clinical samples, however, is an extremely challenging task as there is usually only very limited quantities of material available to perform targeted enrichment approaches. Here, an automated phosphopeptide enrichment protocol using the AssayMap Bravo platform was applied to MM tissues and assessed for performance. The strategy proved to be highly-sensitive, less prone to variability, less laborious than existing techniques and adequate for starting quantities at the microgram level. An Fe(III)-NTA-IMAC-based enrichment workflow was applied to a dilution series of MM tissue lysates. The workflow was efficient in terms of sensitivity, reproducibility and phosphosite localization; and from only 12.5 μg of sample, more than 1,000 phosphopeptides were identified. In addition, from 60 μg of protein material the number of identified phosphoproteins from individual MM samples was comparable to previous reports that used extensive fractionation methods. Our data set included key pathways that are involved in MM progression; such as MAPK, melanocyte development and integrin signaling. Moreover, tissue-specific immunological proteins were identified, that have not been previously observed in the proteome of MM-derived cell lines. In conclusion, this workflow is suitable to study large cohorts of clinical samples that demand automatic and careful handling.
In comparison to other human cancer types, malignant melanoma exhibits the greatest amount of heterogeneity. After DNA-based detection of the BRAF V600E mutation in melanoma patients, targeted ...inhibitor treatment is the current recommendation. This approach, however, does not take the abundance of the therapeutic target, i.e., the B-raf V600E protein, into consideration. As shown by immunohistochemistry, the protein expression profiles of metastatic melanomas clearly reveal the existence of inter- and intra-tumor variability. Nevertheless, the technique is only semi-quantitative. To quantitate the mutant protein there is a fundamental need for more precise techniques that are aimed at defining the currently non-existent link between the levels of the target protein and subsequent drug efficacy. Using cutting-edge mass spectrometry combined with DNA and mRNA sequencing, the mutated B-raf protein within metastatic tumors was quantitated for the first time. B-raf V600E protein analysis revealed a subjacent layer of heterogeneity for mutation-positive metastatic melanomas. These were characterized into two distinct groups with different tumor morphologies, protein profiles and patient clinical outcomes. This study provides evidence that a higher level of expression in the mutated protein is associated with a more aggressive tumor progression. Our study design, comprised of surgical isolation of tumors, histopathological characterization, tissue biobanking, and protein analysis, may enable the eventual delineation of patient responders/non-responders and subsequent therapy for malignant melanoma.
Melanoma of the skin is the sixth most common type of cancer in Europe and accounts for 3.4% of all diagnosed cancers. More alarming is the degree of recurrence that occurs with approximately 20% of ...patients lethally relapsing following treatment. Malignant melanoma is a highly aggressive skin cancer and metastases rapidly extend to the regional lymph nodes (stage 3) and to distal organs (stage 4). Targeted oncotherapy is one of the standard treatment for progressive stage 4 melanoma, and BRAF inhibitors (e.g. vemurafenib, dabrafenib) combined with MEK inhibitor (e.g. trametinib) can effectively counter BRAFV600E-mutated melanomas. Compared to conventional chemotherapy, targeted BRAFV600E inhibition achieves a significantly higher response rate. After a period of cancer control, however, most responsive patients develop resistance to the therapy and lethal progression. The many underlying factors potentially causing resistance to BRAF inhibitors have been extensively studied. Nevertheless, the remaining unsolved clinical questions necessitate alternative research approaches to address the molecular mechanisms underlying metastatic and treatment-resistant melanoma. In broader terms, proteomics can address clinical questions far beyond the reach of genomics, by measuring, i.e. the relative abundance of protein products, post-translational modifications (PTMs), protein localisation, turnover, protein interactions and protein function. More specifically, proteomic analysis of body fluids and tissues in a given medical and clinical setting can aid in the identification of cancer biomarkers and novel therapeutic targets. Achieving this goal requires the development of a robust and reproducible clinical proteomic platform that encompasses automated biobanking of patient samples, tissue sectioning and histological examination, efficient protein extraction, enzymatic digestion, mass spectrometry–based quantitative protein analysis by label-free or labelling technologies and/or enrichment of peptides with specific PTMs. By combining data from, e.g. phosphoproteomics and acetylomics, the protein expression profiles of different melanoma stages can provide a solid framework for understanding the biology and progression of the disease. When complemented by proteogenomics, customised protein sequence databases generated from patient-specific genomic and transcriptomic data aid in interpreting clinical proteomic biomarker data to provide a deeper and more comprehensive molecular characterisation of cellular functions underlying disease progression. In parallel to a streamlined, patient-centric, clinical proteomic pipeline, mass spectrometry–based imaging can aid in interrogating the spatial distribution of drugs and drug metabolites within tissues at single-cell resolution. These developments are an important advancement in studying drug action and efficacy in vivo and will aid in the development of more effective and safer strategies for the treatment of melanoma. A collaborative effort of gargantuan proportions between academia and healthcare professionals has led to the initiation, establishment and development of a cutting-edge cancer research centre with a specialisation in melanoma and lung cancer. The primary research focus of the European Cancer Moonshot Lund Center is to understand the impact that drugs have on cancer at an individualised and personalised level. Simultaneously, the centre increases awareness of the relentless battle against cancer and attracts global interest in the exceptional research performed at the centre.
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•Differentiation analysis of SH-SY5Y cells with iTRAQ strategy is proposed.•Differentiated SH-SY5Y cells are more appropriated as a neuronal model.•Upregulated proteins are mainly ...related to ECM-interaction and apoptosis.•Proteins to explore as differentiation markers: AGRN, EMILIM-1, AIFM, STMN1.
SH-SY5Y neuroblastoma cells are susceptible to differentiation using retinoic acid (RA) and brain-derived neurotrophic factor (BDNF), providing a model of neuronal differentiation. We compared SH-SY5Y cells proteome before and after RA/BDNF treatment using iTRAQ and phosphopeptide enrichment strategies. We identified 5587 proteins, 366 of them with differential abundance. Differentiated cells expressed proteins related to neuronal development, and, undifferentiated cells expressed proteins involved in cell proliferation. Interactive network covered focal adhesion, cytoskeleton dynamics and neurodegenerative diseases processes and regulation of mitogen-activated protein kinase-related signaling pathways; key proteins involved in those processes might be explored as markers for neuronal differentiation.
Zika virus (ZIKV) is a global public health emergency due to its association with microcephaly, Guillain-Barré syndrome, neuropathy, and myelitis in children and adults. A total of 87 countries have ...had evidence of autochthonous mosquito-borne transmission of ZIKV, distributed across four continents, and no antivirus therapy or vaccines are available. Therefore, several strategies have been developed to target the main mosquito vector,
, to reduce the burden of different arboviruses. Among such strategies, the use of the maternally-inherited endosymbiont
has been applied successfully to reduce virus susceptibility and decrease transmission. However, the mechanisms by which
orchestrate resistance to ZIKV infection remain to be elucidated. In this study, we apply isobaric labeling quantitative mass spectrometry (MS)-based proteomics to quantify proteins and identify pathways altered during ZIKV infection;
infection; co-infection with
ZIKV in the
heads and salivary glands. We show that
regulates proteins involved in reactive oxygen species production, regulates humoral immune response, and antioxidant production. The reduction of ZIKV polyprotein in the presence of
in mosquitoes was determined by MS and corroborates the idea that
helps to block ZIKV infections in
The present study offers a rich resource of data that may help to elucidate mechanisms by which
orchestrate resistance to ZIKV infection in
, and represents a step further on the development of new targeted methods to detect and quantify ZIKV and
directly in complex tissues.
Zika is a vector-borne disease caused by an arbovirus (ZIKV) and overwhelmingly transmitted by
Ae. aegypti
. This disease is linked to adverse fetal outcomes, mostly microcephaly in newborns, and ...other clinical aspects such as acute febrile illness and neurologic complications, for example, Guillain-Barré syndrome. One of the most promising strategies to mitigate arbovirus transmission involves releasing
Ae. aegypti
mosquitoes carrying the maternally inherited endosymbiont bacteria
Wolbachia pipientis
. The presence of
Wolbachia
is associated with a reduced susceptibility to arboviruses and a fitness cost in mosquito life-history traits such as fecundity and fertility. However, the mechanisms by which
Wolbachia
influences metabolic pathways leading to differences in egg production remains poorly known. To investigate the impact of coinfections on the reproductive tract of the mosquito, we applied an isobaric labeling-based quantitative proteomic strategy to investigate the influence of
Wolbachia w
Mel and ZIKV infection in
Ae. aegypti
ovaries. To the best of our knowledge, this is the most complete proteome of
Ae. aegypti
ovaries reported so far, with a total of 3913 proteins identified, were also able to quantify 1044
Wolbachia
proteins in complex sample tissue of
Ae. aegypti
ovary. Furthermore, from a total of 480 mosquito proteins modulated in our study, we discuss proteins and pathways altered in
Ae. aegypti
during ZIKV infections,
Wolbachia
infections, coinfection
Wolbachia
/ZIKV, and compared with no infection, focusing on immune and reproductive aspects of
Ae. aegypti
. The modified aspects mainly were related to the immune priming enhancement by
Wolbachia
presence and the modulation of the Juvenile Hormone pathway caused by both microorganism’s infection.
In the advanced stages, malignant melanoma (MM) has a very poor prognosis. Due to tremendous efforts in cancer research over the last 10 years, and the introduction of novel therapies such as ...targeted therapies and immunomodulators, the rather dark horizon of the median survival has dramatically changed from under 1 year to several years. With the advent of proteomics, deep-mining studies can reach low-abundant expression levels. The complexity of the proteome, however, still surpasses the dynamic range capabilities of current analytical techniques. Consequently, many predicted protein products with potential biological functions have not yet been verified in experimental proteomic data. This category of ‘missing proteins’ (MP) is comprised of all proteins that have been predicted but are currently unverified. As part of the initiative launched in 2016 in the USA, the European Cancer Moonshot Center has performed numerous deep proteomics analyses on samples from MM patients. In this study, nine MPs were clearly identified by mass spectrometry in MM metastases. Some MPs significantly correlated with proteins that possess identical PFAM structural domains; and other MPs were significantly associated with cancer-related proteins. This is the first study to our knowledge, where unknown and novel proteins have been annotated in metastatic melanoma tumour tissue.
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