Biology subjects

Moritz, R. L.

Publications and source records attributed to Moritz, R. L..

29 records · Page 2Linked to original sources

bioRxiv2021

Plasmodium falciparum Calcium Dependent Protein Kinase 4 is critical for male gametogenesis and transmission to the mosquito vector

Gametocytes of the malaria parasite Plasmodium are taken up by the mosquito vector with an infectious blood meal, representing a critical stage for parasite transmission. Calcium dependent protein kinases (CDPKs) play key roles in calcium-mediated signaling across the complex life cycle of the parasite. We sought to understand their role in human parasite transmission from the host to the mosquito vector and thus investigated the role of the human-infective parasite Plasmodium falciparum CDPK4 in the parasite life cycle. P. falciparum cdpk4- parasites created by targeted gene deletion showed no effect in blood stage development or gametocyte development. However, cdpk4- parasites showed a severe defect in male gametogenesis and the emergence of flagellated male gametes. To understand the molecular underpinnings of this defect, we performed mass spectrometry-based phosphoproteomic analyses of wild type and Plasmodium falciparum cdpk4- late gametocyte stages, to identify key CDPK4-mediated phosphorylation events that may be important for the regulation of male gametogenesis. We further employed in vitro assays to identify these putative substrates of Plasmodium falciparum CDPK4. This indicated that CDPK4 regulates male gametogenesis by directly or indirectly controlling key essential events such as DNA replication, mRNA translation and cell motility. Taken together, our work demonstrates that PfCDPK4 is a central kinase that regulates exflagellation, and thereby is critical for parasite transmission to the mosquito vector. IMPORTANCETransmission of the malaria parasite to the mosquito vector is critical for the completion of the sexual stage of the parasite life cycle and is dependent on the release of male gametes from the gametocyte body inside the mosquito midgut. In the present study, we demonstrate that PfCDPK4 is critical for male gametogenesis and is involved in phosphorylation of proteins essential for male gamete emergence. Targeting PfCDPK4 and its substrates may provide insights into achieving effective malaria transmission-blocking strategies.

microbiology↗

bioRxiv2020

A wide-ranging Pseudomonas aeruginosa PeptideAtlas build: a useful proteomic resource for a versatile pathogen.

Pseudomonas aeruginosa is an important opportunistic pathogen with high prevalence in nosocomial infections. This microorganism is a good model for understanding biological processes such as the quorum-sensing response, the metabolic integration of virulence, the mechanisms of global regulation of bacterial physiology, and the evolution of antibiotic resistance. Till now, P. aeruginosa proteomic data, although available in several on-line repositories, were dispersed and difficult to access. In the present work, proteomes of the PAO1 strain grown under very different conditions and from diverse cellular compartments have been analyzed and joined to build the Pseudomonas PeptideAtlas. This resource is a comprehensive mass spectrometry-derived peptide and inferred protein database with 71.3% coverage of the total predicted proteome of P. aeruginosa PAO1. This is the highest published coverage among the eight bacterial PeptideAtlas datasets currently available. The proteins in the Pseudomonas PeptideAtlas cover 84% of metabolic proteins, 71% of proteins involved in genetic information processing, 72% of proteins responsible for environmental information processing, more than 80% of proteins related to quorum sensing and biofilm formation, and 81% of proteins responsible for antimicrobial resistance. It exemplifies a necessary tool for targeted proteomics studies, system-wide observations, and cross-species observational studies. Here we describe how this resource was built and some of the physiologically important proteins of this pathogen. SignificancePseudomonas aeruginosa is among the most versatile bacterial pathogens. Studies of its proteome are very important as they can reveal virulence factors and mechanisms of antibiotic resistance. The construction of a proteomic resource such as the PeptideAtlas enables targeted proteomics studies, system-wide observations, and cross-species observational studies.

microbiology↗

bioRxiv2020

A druggable oxidative folding pathway in the endoplasmic reticulum of human malaria parasites

Malaria remains a major global health problem, and there exists a constant need to identify druggable weaknesses in P. falciparum biology. The endoplasmic reticulum (ER) has many essential roles in the asexual lifecycle and may offer new drug targets, but it remains critically understudied. We generated conditional mutants of the putative redox-active, ER chaperone PfJ2, and show that it is essential for parasite survival. Using a redox-active cysteine crosslinker, we identify its substrates to be other mediators of oxidative folding, PfPDI8 and PfPDI11, suggesting a redox-regulatory role for PfJ2. Knockdown of these protein disulfide isomerases in PfJ2 conditional mutants show that PfPDI11 is not essential, while PfPDI8 is essential for asexual growth and may work in a complex with PfJ2 and other ER chaperones. Finally, we show that these redox interactions in the parasite ER are sensitive to small molecule inhibition. Together these data build a model for how oxidative folding occurs in the P. falciparum ER and demonstrate its suitability for antimalarial drug development.

microbiology↗

bioRxiv2020

Platelet Derived Growth Factor Receptor β (PDGFRβ) is a Host Receptor for the human malaria parasite adhesin TRAP

Following their inoculation by the bite of an infected Anopheles mosquito, the malaria parasite sporozoite forms travel from the bite site in the skin into the bloodstream, which transports them to the liver. The thrombospondin-related anonymous protein (TRAP) is a type 1 transmembrane protein that is released from secretory organelles and relocalized on the sporozoite plasma membrane. TRAP is required for sporozoite motility and host infection, and its extracellular portion contains adhesive domains that are predicted to engage host receptors. Here, we identified the human platelet-derived growth factor receptor {beta} (hPDGFR{beta}) as one such protein receptor. Deletion mutants showed that the von Willebrand factor type A and thrombospondin repeat domains of TRAP are both required for optimal binding to hPDGFR{beta}-expressing cells. We also demonstrate that this interaction is conserved in the human-infective parasite Plasmodium vivax, but not the rodent-infective parasite Plasmodium yoelii. We observed expression of hPDGFR{beta} mainly in cells associated with the vasculature suggesting that TRAP:hPDGFR{beta} interaction may play a role in the recognition of blood vessels by invading sporozoites.

microbiology↗

bioRxiv2019

Measurement of organ-specific and acute-phase blood protein levels in early Lyme disease

Lyme disease results from infection of humans with the spirochete Borrelia burgdorferi. The first and most common clinical manifestation is the circular, inflamed skin lesion referred to as erythema migrans; later manifestations result from infections of other body sites. Laboratory diagnosis of Lyme disease can be challenging in patients with erythema migrans because of the time delay in the development of specific diagnostic antibodies against Borrelia. Reliable blood biomarkers for the early diagnosis of Lyme disease in patients with erythema migrans are needed. Here, we performed selected reaction monitoring, a targeted mass spectrometry-based approach, to measure selected proteins that 1) are known to be predominantly expressed in one organ (i.e., organ-specific blood proteins) and whose blood concentrations may change as a result of Lyme disease, or 2) are involved in acute immune responses. In a longitudinal cohort of 40 Lyme disease patients and 20 healthy controls, we identified 10 proteins with significantly altered serum levels in patients at the time of diagnosis, and we also developed a 10-protein panel identified through multivariate analysis. In an independent cohort of patients with erythema migrans, six of these proteins, APOA4, C9, CRP, CST6, PGLYRP2 and S100A9, were confirmed to show significantly altered serum levels in patients at time of presentation. Nine of the 10 proteins from the multivariate panel were also verified in the second cohort. These proteins, primarily innate immune response proteins or proteins specific to liver, skin or white blood cells, may serve as candidate blood biomarkers requiring further validation to aid in the laboratory diagnosis of early Lyme disease.

systems biology↗

bioRxiv2019

Human Proteome Project Mass Spectrometry Data Interpretation Guidelines 3.0

The Human Proteome Organizations (HUPO) Human Proteome Project (HPP) developed Mass Spectrometry (MS) Data Interpretation Guidelines that have been applied since 2016. These guidelines have helped ensure that the emerging draft of the complete human proteome is highly accurate and with low numbers of false-positive protein identifications. Here, we describe an update to these guidelines based on consensus-reaching discussions with the wider HPP community over the past year. The revised 3.0 guidelines address several major and minor identified gaps. We have added guidelines for emerging data independent acquisition (DIA) MS workflows and for use of the new Universal Spectrum Identifier (USI) system being developed by the HUPO Proteomics Standards Initiative (PSI). In addition, we discuss updates to the standard HPP pipeline for collecting MS evidence for all proteins in the HPP, including refinements to minimum evidence. We present a new plan for incorporating MassIVE-KB into the HPP pipeline for the next (HPP 2020) cycle in order to obtain more comprehensive coverage of public MS data sets. The main checklist has been reorganized under headings and subitems and related guidelines have been grouped. In sum, Version 2.1 of the HPP MS Data Interpretation Guidelines has served well and this timely update to version 3.0 will aid the HPP as it approaches its goal of collecting and curating MS evidence of translation and expression for all predicted [~]20,000 human proteins encoded by the human genome.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC=\"FIGDIR/small/733576v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (18K):\norg.highwire.dtl.DTLVardef@8765f2org.highwire.dtl.DTLVardef@136235aorg.highwire.dtl.DTLVardef@14aef76org.highwire.dtl.DTLVardef@af33d2_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2019

PTMProphet: Fast and Accurate Mass Modification Localization for the Trans-Proteomic Pipeline

Spectral matching sequence database search engines commonly used on mass spectrometry-based proteomics experiments excel at identifying peptide sequence ions, and in addition, possible sequence ions carrying post-translational modifications (PTMs), but most do not provide confidence metrics for the exact localization of those PTMs when several possible sites are available. Localization is absolutely required for downstream molecular cell biology analysis of PTM function in vitro and in vivo. Therefore, we developed PTMProphet, a free and open-source software tool integrated into the Trans-Proteomic Pipeline, which reanalyzes identified spectra from any search engine for which pepXML output is available to provide localization confidence to enable appropriate further characterization of biologic events. Localization of any type of mass modification (e.g., phosphorylation) is supported. PTMProphet applies Bayesian mixture models to compute probabilities for each site/peptide spectrum match where a PTM has been identified. These probabilities can be combined to compute a global false localization rate at any threshold to guide downstream analysis. We describe the PTMProphet tool, its underlying algorithms and demonstrate its performance on ground-truth synthetic peptide reference datasets, one previously published small dataset, one new larger dataset, and also on a previously published phospho-enriched dataset where the correct sites of modification are unknown. Data have been deposited to ProteomeXchange with identifier PXD013210.\n\nO_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

bioinformatics↗

bioRxiv2019

Extensive Transcriptional and Translational Regulation Occur During the Maturation of Malaria Parasite Sporozoites

Plasmodium sporozoites are transmitted from an infected mosquito to mammals in which they infect the liver. The infectivity profile of sporozoites changes as they egress from oocysts on the mosquito midgut into the hemocoel, and then invade the salivary glands, where they maintain a poised and infectious state until transmission occurs. Upon transmission, the sporozoite must then navigate the host skin, vasculature, and liver. All of these feats require distinct repertoires of proteins and capabilities that are coordinated in an appropriate temporal manner. Here, we report the comprehensive and dynamic transcriptomes and proteomes of both oocyst sporozoite and salivary gland sporozoite stages in both rodent-infectious Plasmodium yoelii parasites and human-infectious Plasmodium falciparum parasites. These data robustly define mRNAs and proteins that are Upregulated in Oocyst Sporozoites (UOS) or Upregulated in Infectious Sporozoites (UIS), which include critical gene products for sporozoite functions, as well as many of unknown importance that are similarly regulated. Moreover, we found that Plasmodium uses two overlapping, extensive, and independent programs of translational repression across sporozoite maturation to temporally regulate specific genes necessary to successfully navigate the mosquito vector and mammalian host environments. Finally, gene-specific validation experiments of selected, translationally repressed transcripts in P. yoelii confirmed the interpretations of the global transcriptomic and proteomic datasets. Together, these data indicate that two waves of translational repression are implemented and relieved at different times in sporozoite maturation to promote its successful life cycle progression.

microbiology↗

bioRxiv2019

Proteomic Analysis of Plasmodium Merosomes: The Link Between Liver and Blood Stages in Malaria

The pre-erythrocytic liver stage of the malaria parasite, comprising sporozoites and the liver stages into which they develop, remains one of the least understood parts of the lifecycle, in part owing to the low numbers of parasites. Nonetheless, it is recognized as an important target for anti-malarial drugs and vaccines. Here we provide the first proteomic analysis of merosomes, which define the final phase of the liver stage and are responsible for initiating the blood stage of infection. We identify a total of 1879 parasite proteins, and a core set of 1188 proteins quantitatively detected in every biological replicate, providing an extensive picture of the protein repertoire of this stage. This unique dataset will allow us to explore key questions about the biology of merosomes and hepatic merozoites.\n\nHighlightsO_LIFirst proteome of the merosome stage of malaria parasites\nC_LIO_LIQuantitative detection of 1188 parasite proteins across 3 biological replicates\nC_LIO_LIComparison to blood stage proteomes identifies shared and unique proteins\nC_LIO_LIDiscovery of cleaved PEXEL motifs highlights liver stage protein export\nC_LI\n\nIn BriefThe merosome stage that links malaria liver and blood stage infection is poorly understood. Here we provide the first proteome of this life cycle stage using the Plasmodium berghei rodent malaria model.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=27 SRC=\"FIGDIR/small/580266_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (11K):\norg.highwire.dtl.DTLVardef@5bd8b1org.highwire.dtl.DTLVardef@1ef4fb9org.highwire.dtl.DTLVardef@5b3833org.highwire.dtl.DTLVardef@c69c73_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2019

Luminal Calcification of the Microvasculature in Fetuin-A Deficient Mice Leads to Premature Aging and Death

ObjectiveThe plasma protein fetuin-A mediates the formation of protein-mineral colloids known as calciprotein particles (CPP) - rapid clearance of these CPP by the reticuloendothelial system prevents errant mineral precipitation and therefore ectopic mineralization (calcification). The mutant mouse strain D2,Ahsg-/- combines fetuin-A deficiency with the mineralization-prone DBA/2 genetic background, having a particularly severe compound phenotype of microvascular and soft tissue mineralization. Here we studied mechanisms leading to soft tissue mineralization, organ damage and premature aging in these mice.\n\nApproach and ResultsWe analyzed mice longitudinally by echocardiography, X-ray-computed tomography, analytical electron microscopy, histology, mass spectrometry proteomics, and genome-wide microarray-based expression analyses of D2 wildtype and Ahsg-/- mice.\n\nFetuin-A deficient mice had calcified lesions in myocardium, lung, brown adipose tissue, reproductive organs, spleen, pancreas, kidney and the skin, associated with reduced growth, cardiac output and premature aging. Importantly, early stage calcified lesions presented in the lumen of the microvasculature suggesting precipitation of mineral containing complexes from the fluid phase of blood. Genome-wide expression analysis of calcified lesions and surrounding (not calcified) tissue, together with morphological observations, indicated that the ectopic mineralization was not associated with osteochondrogenic cell differentiation, but rather with thrombosis and fibrosis.\n\nConclusionsCollectively, these results demonstrate that pathological mineralization can start by intravascular mineral deposition causing microvasculopathy, which impacts on growth, organ function and survival. Our study underscores the importance of fetuin-A and related systemic regulators of mineralized matrix metabolism to prevent cardiovascular disease, especially in dysregulated mineral homeostasis.

physiology↗

bioRxiv2018

Cross-linking/Mass Spectrometry: A Community-Wide, Comparative Study Towards Establishing Best Practice Guidelines

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has largely increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats that are generally accepted in the field and that have shown to lead to high-quality results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. From the results obtained, common protocols will be established. Our study serves as basis for establishing best practice guidelines in the field for conducting cross-linking experiments, performing data analysis, and reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.

biochemistry↗