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Paulo, Joao

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Paulo

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Joao

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Paulo, Joao
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    Quantitative Temporal in Vivo Proteomics Deciphers the Transition of Virus-Driven Myeloid Cells into M2 Macrophages
    (American Chemical Society, 2017) Clements, Derek R.; Murphy, John Patrick; Sterea, Andra; Kennedy, Barry E.; Kim, Youra; Helson, Erin; Almasi, Shekoufeh; Holay, Namit; Konda, Prathyusha; Paulo, Joao; Sharif, Tanveer; Lee, Patrick W.; Weekes, Michael P.; Gygi, Steven; Gujar, Shashi
    Myeloid cells play a central role in the context of viral eradication, yet precisely how these cells differentiate throughout the course of acute infections is poorly understood. In this study, we have developed a novel quantitative temporal in vivo proteomics (QTiPs) platform to capture proteomic signatures of temporally transitioning virus-driven myeloid cells directly in situ, thus taking into consideration host–virus interactions throughout the course of an infection. QTiPs, in combination with phenotypic, functional, and metabolic analyses, elucidated a pivotal role for inflammatory CD11b+, Ly6G–, Ly6Chigh-low cells in antiviral immune response and viral clearance. Most importantly, the time-resolved QTiPs data set showed the transition of CD11b+, Ly6G–, Ly6Chigh-low cells into M2-like macrophages, which displayed increased antigen-presentation capacities and bioenergetic demands late in infection. We elucidated the pivotal role of myeloid cells in virus clearance and show how these cells phenotypically, functionally, and metabolically undergo a timely transition from inflammatory to M2-like macrophages in vivo. With respect to the growing appreciation for in vivo examination of viral–host interactions and for the role of myeloid cells, this study elucidates the use of quantitative proteomics to reveal the role and response of distinct immune cell populations throughout the course of virus infection.
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    Plasmodium falciparum CRK4 directs continuous rounds of DNA replication during schizogony
    (2017) Ganter, Markus; Goldberg, Jonathan; Dvorin, Jeffrey; Paulo, Joao; King, Jonas G.; Tripathi, Abhai K.; Paul, Aditya; Yang, Jing; Coppens, Isabelle; Jiang, Rays H.Y.; Elsworth, Brendan; Baker, David A.; Dinglasan, Rhoel R.; Gygi, Steven; Duraisingh, Manoj
    Plasmodium parasites, the causative agents of malaria, have evolved a unique cell division cycle in the clinically relevant asexual blood-stage of infection1. DNA replication commences approximately halfway through the intracellular development following invasion and parasite growth. The schizont stage is associated with multiple rounds of DNA replication and nuclear division without cytokinesis resulting in a multinucleated cell. Nuclei divide asynchronously through schizogony, with only the final round of DNA replication and segregation being synchronous and coordinated with daughter cell assembly2,3. However, the control mechanisms for this divergent mode of replication are unknown. Here we show that the Plasmodium-specific kinase PfCRK4 is a key cell cycle regulator that orchestrates the multiple rounds of DNA replication throughout schizogony in P. falciparum. PfCRK4 depletion led to a complete block in nuclear division and profoundly inhibited DNA replication. Quantitative phosphoproteomic profiling identified a set of PfCRK4-regulated phosphoproteins with greatest functional similarity to CDK2 substrates, particularly proteins involved in origin of replication firing. PfCRK4 was required for the initial and subsequent rounds of DNA replication during schizogony, and in addition was essential for development in the mosquito vector. Our results identified an essential S phase promoting factor of the unconventional P. falciparum cell cycle. PfCRK4 is required for both a prolonged period of the intraerythrocytic blood-stage of malaria infection, as well as for transmission, revealing a broad window for PfCRK4-targeted chemotherapeutics.
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    Compositional Proteomics: Effects of Spatial Constraints on Protein Quantification Utilizing Isobaric Tags
    (American Chemical Society, 2017) O’Brien, Jonathon J.; O’Connell, Jeremy D.; Paulo, Joao; Thakurta, Sanjukta; Rose, Christopher M.; Weekes, Michael P.; Huttlin, Edward; Gygi, Steven
    Mass spectrometry (MS) has become an accessible tool for whole proteome quantitation with the ability to characterize protein expression across thousands of proteins within a single experiment. A subset of MS quantification methods (e.g., SILAC and label-free) monitor the relative intensity of intact peptides, where thousands of measurements can be made from a single mass spectrum. An alternative approach, isobaric labeling, enables precise quantification of multiple samples simultaneously through unique and sample specific mass reporter ions. Consequently, in a single scan, the quantitative signal comes from a limited number of spectral features (≤11). The signal observed for these features is constrained by automatic gain control, forcing codependence of concurrent signals. The study of constrained outcomes primarily belongs to the field of compositional data analysis. We show experimentally that isobaric tag proteomics data are inherently compositional and highlight the implications for data analysis and interpretation. We present a new statistical model and accompanying software that improves estimation accuracy and the ability to detect changes in protein abundance. Finally, we demonstrate a unique compositional effect on proteins with infinite changes. We conclude that many infinite changes will appear small and that the magnitude of these estimates is highly dependent on experimental design.
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    Unique roles for histone H3K9me states in RNAi and heritable silencing of transcription
    (2017) Jih, Gloria; Iglesias, Nahid; Currie, Mark; Bhanu, Natarajan V.; Paulo, Joao; Gygi, Steven; Garcia, Benjamin A.; Moazed, Danesh
    Heterochromatic DNA domains play important roles in regulation of gene expression and maintenance of genome stability by silencing repetitive DNA elements and transposons. From fission yeast to mammals, heterochromatin assembly at DNA repeats involves the activity of small noncoding RNAs (sRNAs) associated with the RNA interference (RNAi) pathway1–9. Typically, sRNAs, originating from long noncoding RNAs, guide Argonaute-containing effector complexes to complementary nascent RNAs to initiate histone H3 lysine 9 di- and tri-methylation (H3K9me2 and H3K9me3, respectively) and heterochromatin formation10–17. H3K9me is in turn required for recruitment of RNAi to chromatin to promote sRNA amplification11,15,18. Yet, how heterochromatin formation, which silences transcription, can proceed by a co-transcriptional mechanism that also promotes sRNA generation remains paradoxical. Here, using Clr4, the fission yeast S. pombe homolog of mammalian SUV39H H3K9 methyltransferases, we designed active site mutations that block H3K9me3, but allow H3K9me2 catalysis. We show that H3K9me2 defines a functionally distinct heterochromatin state that is sufficient for RNAi-dependent co-transcriptional gene silencing (CTGS) at pericentromeric DNA repeats. Unlike H3K9me3 domains, which are transcriptionally silent, H3K9me2 domains are transcriptionally active, contain modifications associated with euchromatic transcription, and couple RNAi-mediated transcript degradation to the establishment of H3K9me domains. The two H3K9me states recruit reader proteins with different efficiencies, explaining their different downstream silencing functions. Furthermore, transition from H3K9me2 to H3K9me3 is required for RNAi-independent epigenetic inheritance of H3K9me domains. Our findings demonstrate that H3K9me2 and H3K9me3 define functionally distinct chromatin states and uncover a mechanism for formation of transcriptionally permissive heterochromatin that is compatible with its broadly conserved role in sRNA-mediated genome defense.
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    Ubiquilin1 promotes antigen-receptor mediated proliferation by eliminating mislocalized mitochondrial proteins
    (eLife Sciences Publications, Ltd, 2017) Whiteley, Alexandra; Prado, Miguel A; Peng, Ivan; Abbas, Alexander R; Haley, Benjamin; Paulo, Joao; Reichelt, Mike; Katakam, Anand; Sagolla, Meredith; Modrusan, Zora; Lee, Dong Yun; Roose-Girma, Merone; Kirkpatrick, Donald S; McKenzie, Brent S; Gygi, Steven; Finley, Daniel; Brown, Eric J
    Ubiquilins (Ubqlns) are a family of ubiquitin receptors that promote the delivery of hydrophobic and aggregated ubiquitinated proteins to the proteasome for degradation. We carried out a proteomic analysis of a B cell lymphoma-derived cell line, BJAB, that requires UBQLN1 for survival to identify UBQLN1 client proteins. When UBQLN1 expression was acutely inhibited, 120 mitochondrial proteins were enriched in the cytoplasm, suggesting that the accumulation of mitochondrial client proteins in the absence of UBQLN1 is cytostatic. Using a Ubqln1−/− mouse strain, we found that B cell receptor (BCR) ligation of Ubqln1−/− B cells led to a defect in cell cycle entry. As in BJAB cells, mitochondrial proteins accumulated in BCR-stimulated cells, leading to protein synthesis inhibition and cell cycle block. Thus, UBQLN1 plays an important role in clearing mislocalized mitochondrial proteins upon cell stimulation, and its absence leads to suppression of protein synthesis and cell cycle arrest.
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    Screening Preeclamptic Cord Plasma for Proteins Associated with Decreased Breast Cancer Susceptibility
    (Elsevier, 2013) Low, Hoi Pang; Tiwari, Ashutosh; Janjanam, Jagadeesh; Qiu, Li; Chang, Chien-I; Strohsnitter, William C.; Norwitz, Errol R.; Tam, Sun W.; Evans, James E.; Green, Karin M.; Paulo, Joao; Lambe, Mats; Hsieh, Chung-Cheng
    Preeclampsia, a complication of pregnancy characterized by hypertension and proteinuria, has been found to reduce the subsequent risk for breast cancer in female offspring. As this protective effect could be due to exposure to preeclampsia-specific proteins during intrauterine life, the proteomic profiles of umbilical cord blood plasma between preeclamptic and normotensive pregnancies were compared. Umbilical cord plasma samples, depleted of 14 abundant proteins, were subjected to proteomic analysis using the quantitative method of nanoACQUITY ultra performance liquid chromatography–mass spectrometry with elevated energy mode of acquisitionE (NanoUPLC-MSE). Sixty-nine differentially expressed proteins were identified, of which 15 and 6 proteins were only detected in preeclamptic and normotensive pregnancies, respectively. Additionally, expression of 8 proteins (gelsolin, complement C5, keratin type I cytoskeletal 10, pigment epithelium-derived factor, complement factor B, complement component C7, hemoglobin subunit gamma-2 and alpha-fetoprotein) were up-regulated in preeclampsia with a fold change of ⩾2.0 when compared to normotensive pregnancies. The identification of alpha-fetoprotein in preeclamptic umbilical cord blood plasma supported the validity of this screen as alpha-fetoprotein has anti-estrogenic properties and has previously been linked to preeclampsia as well as a reduced breast cancer risk. The findings of this pilot study may provide new insights into the mechanistic link between preeclampsia and potentially reduced breast cancer susceptibility in adult life.
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    QIL1 is a novel mitochondrial protein required for MICOS complex stability and cristae morphology
    (eLife Sciences Publications, Ltd, 2015) Guarani, Virginia; McNeill, Elizabeth; Paulo, Joao; Huttlin, Edward; Fröhlich, Florian; Gygi, Steven; Van Vactor, David; Harper, J Wade
    The mitochondrial contact site and cristae junction (CJ) organizing system (MICOS) dynamically regulate mitochondrial membrane architecture. Through systematic proteomic analysis of human MICOS, we identified QIL1 (C19orf70) as a novel conserved MICOS subunit. QIL1 depletion disrupted CJ structure in cultured human cells and in Drosophila muscle and neuronal cells in vivo. In human cells, mitochondrial disruption correlated with impaired respiration. Moreover, increased mitochondrial fragmentation was observed upon QIL1 depletion in flies. Using quantitative proteomics, we show that loss of QIL1 resulted in MICOS disassembly with the accumulation of a MIC60-MIC19-MIC25 sub-complex and degradation of MIC10, MIC26, and MIC27. Additionally, we demonstrated that in QIL1-depleted cells, overexpressed MIC10 fails to significantly restore its interaction with other MICOS subunits and SAMM50. Collectively, our work uncovers a previously unrecognized subunit of the MICOS complex, necessary for CJ integrity, cristae morphology, and mitochondrial function and provides a resource for further analysis of MICOS architecture. DOI: http://dx.doi.org/10.7554/eLife.06265.001
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    A Temporal Proteomic Map of Epstein-Barr Virus Lytic Replication in B Cells
    (Cell Press, 2017) Ersing, Ina; Nobre, Luis; Wang, Liang Wei; Soday, Lior; Ma, Yijie; Paulo, Joao; Narita, Yohei; Ashbaugh, Camille W.; Jiang, Chang; Grayson, Nicholas E.; Kieff, Elliott; Gygi, Steven; Weekes, Michael P.; Gewurz, Benjamin
    Summary Epstein-Barr virus (EBV) replication contributes to multiple human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, B cell lymphomas, and oral hairy leukoplakia. We performed systematic quantitative analyses of temporal changes in host and EBV proteins during lytic replication to gain insights into virus-host interactions, using conditional Burkitt lymphoma models of type I and II EBV infection. We quantified profiles of >8,000 cellular and 69 EBV proteins, including >500 plasma membrane proteins, providing temporal views of the lytic B cell proteome and EBV virome. Our approach revealed EBV-induced remodeling of cell cycle, innate and adaptive immune pathways, including upregulation of the complement cascade and proteasomal degradation of the B cell receptor complex, conserved between EBV types I and II. Cross-comparison with proteomic analyses of human cytomegalovirus infection and of a Kaposi-sarcoma-associated herpesvirus immunoevasin identified host factors targeted by multiple herpesviruses. Our results provide an important resource for studies of EBV replication.
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    Control of immune ligands by members of a cytomegalovirus gene expansion suppresses natural killer cell activation
    (eLife Sciences Publications, Ltd, 2017) Fielding, Ceri A; Weekes, Michael P; Nobre, Luis V; Ruckova, Eva; Wilkie, Gavin S; Paulo, Joao; Chang, Chiwen; Suárez, Nicolás M; Davies, James A; Antrobus, Robin; Stanton, Richard J; Aicheler, Rebecca J; Nichols, Hester; Vojtesek, Borek; Trowsdale, John; Davison, Andrew J; Gygi, Steven; Tomasec, Peter; Lehner, Paul J; Wilkinson, Gavin W G
    The human cytomegalovirus (HCMV) US12 family consists of ten sequentially arranged genes (US12-21) with poorly characterized function. We now identify novel natural killer (NK) cell evasion functions for four members: US12, US14, US18 and US20. Using a systematic multiplexed proteomics approach to quantify ~1300 cell surface and ~7200 whole cell proteins, we demonstrate that the US12 family selectively targets plasma membrane proteins and plays key roles in regulating NK ligands, adhesion molecules and cytokine receptors. US18 and US20 work in concert to suppress cell surface expression of the critical NKp30 ligand B7-H6 thus inhibiting NK cell activation. The US12 family is therefore identified as a major new hub of immune regulation. DOI: http://dx.doi.org/10.7554/eLife.22206.001
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    Role of the B Allele of Influenza A Virus Segment 8 in Setting Mammalian Host Range and Pathogenicity
    (American Society for Microbiology, 2016) Turnbull, Matthew L.; Wise, Helen M.; Nicol, Marlynne Q.; Smith, Nikki; Dunfee, Rebecca L.; Beard, Philippa M.; Jagger, Brett W.; Ligertwood, Yvonne; Hardisty, Gareth R.; Xiao, Haixia; Benton, Donald J.; Coburn, Alice M.; Paulo, Joao; Gygi, Steven; McCauley, John W.; Taubenberger, Jeffery K.; Lycett, Samantha J.; Weekes, Michael P.; Dutia, Bernadette M.; Digard, Paul
    ABSTRACT Two alleles of segment 8 (NS) circulate in nonchiropteran influenza A viruses. The A allele is found in avian and mammalian viruses, but the B allele is viewed as being almost exclusively found in avian viruses. This might reflect the fact that one or both of its encoded proteins (NS1 and NEP) are maladapted for replication in mammalian hosts. To test this, a number of clade A and B avian virus-derived NS segments were introduced into human H1N1 and H3N2 viruses. In no case was the peak virus titer substantially reduced following infection of various mammalian cell types. Exemplar reassortant viruses also replicated to similar titers in mice, although mice infected with viruses with the avian virus-derived segment 8s had reduced weight loss compared to that achieved in mice infected with the A/Puerto Rico/8/1934 (H1N1) parent. In vitro, the viruses coped similarly with type I interferons. Temporal proteomics analysis of cellular responses to infection showed that the avian virus-derived NS segments provoked lower levels of expression of interferon-stimulated genes in cells than wild type-derived NS segments. Thus, neither the A nor the B allele of avian virus-derived NS segments necessarily attenuates virus replication in a mammalian host, although the alleles can attenuate disease. Phylogenetic analyses identified 32 independent incursions of an avian virus-derived A allele into mammals, whereas 6 introductions of a B allele were identified. However, A-allele isolates from birds outnumbered B-allele isolates, and the relative rates of Aves-to-Mammalia transmission were not significantly different. We conclude that while the introduction of an avian virus segment 8 into mammals is a relatively rare event, the dogma of the B allele being especially restricted is misleading, with implications in the assessment of the pandemic potential of avian influenza viruses. IMPORTANCE: Influenza A virus (IAV) can adapt to poultry and mammalian species, inflicting a great socioeconomic burden on farming and health care sectors. Host adaptation likely involves multiple viral factors. Here, we investigated the role of IAV segment 8. Segment 8 has evolved into two distinct clades: the A and B alleles. The B-allele genes have previously been suggested to be restricted to avian virus species. We introduced a selection of avian virus A- and B-allele segment 8s into human H1N1 and H3N2 virus backgrounds and found that these reassortant viruses were fully competent in mammalian host systems. We also analyzed the currently available public data on the segment 8 gene distribution and found surprisingly little evidence for specific avian host restriction of the B-clade segment. We conclude that B-allele segment 8 genes are, in fact, capable of supporting infection in mammals and that they should be considered during the assessment of the pandemic risk of zoonotic influenza A viruses.