Publications by authors named "Manor Askenazi"

37 Publications

Proteomics and Transcriptomics of the Hippocampus and Cortex in SUDEP and High-Risk SUDEP Patients.

Neurology 2021 Apr 28. Epub 2021 Apr 28.

Comprehensive Epilepsy Center, NYU School of Medicine, New York, NY, USA.

Objective: To identify the molecular signaling pathways underlying sudden unexpected death in epilepsy (SUDEP) and high-risk SUDEP compared to epilepsy control patients.

Methods: For proteomics analyses, we evaluated the hippocampus and frontal cortex from microdissected post-mortem brain tissue of 12 SUDEP and 14 non-SUDEP epilepsy patients. For transcriptomics analyses, we evaluated hippocampus and temporal cortex surgical brain tissue from mesial temporal lobe epilepsy (MTLE) patients: 6 low-risk and 8 high-risk SUDEP as determined by a short (< 50 seconds) or prolonged (≥ 50 seconds) postictal generalized EEG suppression (PGES) that may indicate severely depressed brain activity impairing respiration, arousal, and protective reflexes.

Results: In autopsy hippocampus and cortex, we observed no proteomic differences between SUDEP and non-SUDEP epilepsy patients, contrasting with our previously reported robust differences between epilepsy and non-epilepsy control patients. Transcriptomics in hippocampus and cortex from surgical epilepsy patients segregated by PGES identified 55 differentially expressed genes (37 protein-coding, 15 lncRNAs, three pending) in hippocampus.

Conclusion: The SUDEP proteome and high-risk SUDEP transcriptome were similar to other epilepsy patients in hippocampus and frontal cortex, consistent with diverse epilepsy syndromes and comorbidities associated with SUDEP. Studies with larger cohorts and different epilepsy syndromes, as well as additional anatomic regions may identify molecular mechanisms of SUDEP.
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http://dx.doi.org/10.1212/WNL.0000000000011999DOI Listing
April 2021

Mitochondrial STAT3 regulates antioxidant gene expression through complex I-derived NAD in triple negative breast cancer.

Mol Oncol 2021 May 10;15(5):1432-1449. Epub 2021 Apr 10.

Department of Pathology and NYU Perlmutter Cancer Center, NYU School of Medicine, New York, NY, USA.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor with roles in inflammation and tumorigenicity. A fraction of STAT3 localizes in mitochondria, where it augments tumorigenesis via regulation of mitochondrial functions, including modulation of respiration and redox status. We show a novel mechanism for mitochondrial STAT3 regulation of redox homeostasis in triple-negative breast cancer cells. Loss of STAT3 diminished complex I dehydrogenase activity and impaired NAD+ regeneration, leading to impaired expression of glutathione biosynthetic genes and other antioxidant genes. Expressing mitochondrially restricted STAT3 or replenishment of the cellular NAD pool restored antioxidant gene expression, as did complementation of the NADH dehydrogenase activity by expression of the STAT3-independent yeast dehydrogenase, NDI1. These NAD-regulated processes contributed to malignant phenotypes by promoting clonal cell growth and migration. Proximity interaction and protein pull-down assays identified three components of complex I that associated with mitochondrial STAT3, providing a potential mechanistic basis for how mitochondrial STAT3 affects complex I activity. Our data document a novel mechanism through which mitochondrial STAT3 indirectly controls antioxidant gene regulation through a retrograde NAD+ signal that is modulated by complex I dehydrogenase activity.
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http://dx.doi.org/10.1002/1878-0261.12928DOI Listing
May 2021

Evolution of neuroinflammation across the lifespan of individuals with Down syndrome.

Brain 2020 12;143(12):3653-3671

Department of Anatomy and Cell Biology, McGill University, Montreal, Canada.

Epidemiological and experimental studies suggest that a disease-aggravating neuroinflammatory process is present at preclinical stages of Alzheimer's disease. Given that individuals with Down syndrome are at increased genetic risk of Alzheimer's disease and therefore develop the spectrum of Alzheimer's neuropathology in a uniform manner, they constitute an important population to study the evolution of neuroinflammation across the Alzheimer's continuum. Therefore, in this cross-sectional study, we characterized the brain inflammatory profile across the lifespan of individuals with Down syndrome. Microglial morphology and inflammatory cytokine expression were analysed by immunohistochemistry and electrochemiluminescent-based immunoassays in the frontal cortex from foetuses to adults with Down syndrome and control subjects (16 gestational weeks to 64 years), totalling 127 cases. Cytokine expression in mixed foetal primary cultures and hippocampus of adults with Down syndrome, as well as the effects of sex on cytokine expression were also analysed. A higher microglial soma size-to-process length ratio was observed in the frontal cortex of children and young adults with Down syndrome before the development of full-blown Alzheimer's pathology. Moreover, young adults with Down syndrome also displayed increased numbers of rod-like microglia. Increased levels of interleukin-8 and interleukin-10 were observed in children with Down syndrome (1-10 years; Down syndrome n = 5, controls n = 10) and higher levels of interleukin-1β, interleukin-1α, interleukin-6, interleukin-8, interleukin-10, interleukin-15, eotaxin-3, interferon gamma-induced protein 10, macrophage-derived chemokine, and macrophage inflammatory protein-beta, were found in young adults with Down syndrome compared to euploid cases (13-25 years, Down syndrome n = 6, controls n = 24). Increased cytokine expression was also found in the conditioned media of mixed cortical primary cultures from second trimester foetuses with Down syndrome (Down syndrome n = 7, controls n = 7). Older adults with Down syndrome (39-68 years, Down syndrome n = 22, controls n = 16) displayed reduced levels of interleukin-10, interleukin-12p40, interferon-gamma and tumour necrosis factor-alpha. Microglia displayed larger somas and shorter processes. Moreover, an increase in dystrophic microglia and rod-like microglia aligning to neurons harbouring tau pathology were also observed. Sex stratification analyses revealed that females with Down syndrome had increased interleukin-6 and interleukin-8 levels compared to males with Down syndrome. Finally, multivariate projection methods identified specific cytokine patterns among individuals with Down syndrome. Our findings indicate the presence of an early and evolving neuroinflammatory phenotype across the lifespan in Down syndrome, a knowledge that is relevant for the discovery of stage-specific targets and for the design of possible anti-inflammatory trials against Alzheimer's disease in this population.
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http://dx.doi.org/10.1093/brain/awaa326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7805813PMC
December 2020

PRiSM: A prototype for exhaustive, restriction-free database searching for mass spectrometry-based identification.

Rapid Commun Mass Spectrom 2020 Oct 2:e8962. Epub 2020 Oct 2.

Universiteit Hasselt, Data Science Institute (DSI), Theoretical Physics, Diepenbeek, Belgium.

Rationale: The current methods for identifying peptides in mass spectral product ion data still struggle to do so for the majority of spectra. Based on the experimental setup and other assumptions, such methods restrict the search space to speed up computations, but at the cost of creating blind spots. The proteomics community would greatly benefit from a method that is capable of covering the entire search space without using any restrictions, thus establishing a baseline for identification.

Methods: We conceived the "mass pattern paradigm" (MPP) that enables the creation of such an identification method, and we implemented it into a prototype database search engine "PRiSM" (PRotein-Spectrum Matching). We then assessed its operational characteristics by applying it to publicly available high-precision mass spectra of low and high identification difficulty. We used those characteristics to gain theoretical insights into trade-offs between sensitivity and speed when trying to establish a baseline for identification.

Results: Of 100 low difficulty spectra, PRiSM and SEQUEST agree on 84 identifications (of which 75 are statistically significant). Of 15 of 100 spectra not identified in a previous study (using SEQUEST), 13 are considered reliable after visual inspection and represent 3 proteins (out of 9 in total) not detected previously.

Conclusions: Despite leaving noise intact, the simple PRiSM prototype can make statistically reliable identifications, while controlling the false discovery rate by fitting a null distribution. It also identifies some spectra previously unidentifiable in an "extremely open" SEQUEST search, paving the way to establishing a baseline for identification in proteomics.
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http://dx.doi.org/10.1002/rcm.8962DOI Listing
October 2020

Phosphorylated tau interactome in the human Alzheimer's disease brain.

Brain 2020 09;143(9):2803-2817

Centre for Cognitive Neurology, Department of Neurology, New York University School of Medicine, New York, NY, USA.

Accumulation of phosphorylated tau is a key pathological feature of Alzheimer's disease. Phosphorylated tau accumulation causes synaptic impairment, neuronal dysfunction and formation of neurofibrillary tangles. The pathological actions of phosphorylated tau are mediated by surrounding neuronal proteins; however, a comprehensive understanding of the proteins that phosphorylated tau interacts with in Alzheimer's disease is surprisingly limited. Therefore, the aim of this study was to determine the phosphorylated tau interactome. To this end, we used two complementary proteomics approaches: (i) quantitative proteomics was performed on neurofibrillary tangles microdissected from patients with advanced Alzheimer's disease; and (ii) affinity purification-mass spectrometry was used to identify which of these proteins specifically bound to phosphorylated tau. We identified 542 proteins in neurofibrillary tangles. This included the abundant detection of many proteins known to be present in neurofibrillary tangles such as tau, ubiquitin, neurofilament proteins and apolipoprotein E. Affinity purification-mass spectrometry confirmed that 75 proteins present in neurofibrillary tangles interacted with PHF1-immunoreactive phosphorylated tau. Twenty-nine of these proteins have been previously associated with phosphorylated tau, therefore validating our proteomic approach. More importantly, 34 proteins had previously been associated with total tau, but not yet linked directly to phosphorylated tau (e.g. synaptic protein VAMP2, vacuolar-ATPase subunit ATP6V0D1); therefore, we provide new evidence that they directly interact with phosphorylated tau in Alzheimer's disease. In addition, we also identified 12 novel proteins, not previously known to be physiologically or pathologically associated with tau (e.g. RNA binding protein HNRNPA1). Network analysis showed that the phosphorylated tau interactome was enriched in proteins involved in the protein ubiquitination pathway and phagosome maturation. Importantly, we were able to pinpoint specific proteins that phosphorylated tau interacts with in these pathways for the first time, therefore providing novel potential pathogenic mechanisms that can be explored in future studies. Combined, our results reveal new potential drug targets for the treatment of tauopathies and provide insight into how phosphorylated tau mediates its toxicity in Alzheimer's disease.
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http://dx.doi.org/10.1093/brain/awaa223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526722PMC
September 2020

Visualizing the agreement of peptide assignments between different search engines.

J Mass Spectrom 2020 Aug 3;55(8):e4471. Epub 2019 Dec 3.

Interuniversity Institute of Biostatistics and Statistical Bioinformatics, Hasselt University, Hasselt, Belgium.

There is a trend in the analysis of shotgun proteomics data that aims to combine information from multiple search engines to increase the number of peptide annotations in an experiment. Typically, the degree of search engine complementarity and search engine agreement is visually illustrated by means of Venn diagrams that present the findings of a database search on the level of the nonredundant peptide annotations. We argue this practice to be not fit-for-purpose since the diagrams do not take into account and often conceal the information on complementarity and agreement at the level of the spectrum identification. We promote a new type of visualization that provides insight on the peptide sequence agreement at the level of the peptide-spectrum match (PSM) as a measure of consensus between two search engines with nominal outcomes. We applied the visualizations and percentage sequence agreement to an in-house data set of our benchmark organism, Caenorhabditis elegans, and illustrated that when assessing the agreement between search engine, one should disentangle the notion of PSM confidence and PSM identity. The visualizations presented in this manuscript provide a more informative assessment of pairs of search engines and are made available as an R function in the Supporting Information.
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http://dx.doi.org/10.1002/jms.4471DOI Listing
August 2020

Nrf2 Activation Promotes Lung Cancer Metastasis by Inhibiting the Degradation of Bach1.

Cell 2019 07 27;178(2):316-329.e18. Epub 2019 Jun 27.

Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA; Perlmutter NYU Cancer Center, New York University School of Medicine, New York, NY 10016, USA; Howard Hughes Medical Institute, New York University School of Medicine, New York, NY 10016, USA. Electronic address:

Approximately 30% of human lung cancers acquire mutations in either Keap1 or Nfe2l2, resulting in the stabilization of Nrf2, the Nfe2l2 gene product, which controls oxidative homeostasis. Here, we show that heme triggers the degradation of Bach1, a pro-metastatic transcription factor, by promoting its interaction with the ubiquitin ligase Fbxo22. Nrf2 accumulation in lung cancers causes the stabilization of Bach1 by inducing Ho1, the enzyme catabolizing heme. In mouse models of lung cancers, loss of Keap1 or Fbxo22 induces metastasis in a Bach1-dependent manner. Pharmacological inhibition of Ho1 suppresses metastasis in a Fbxo22-dependent manner. Human metastatic lung cancer display high levels of Ho1 and Bach1. Bach1 transcriptional signature is associated with poor survival and metastasis in lung cancer patients. We propose that Nrf2 activates a metastatic program by inhibiting the heme- and Fbxo22-mediated degradation of Bach1, and that Ho1 inhibitors represent an effective therapeutic strategy to prevent lung cancer metastasis.
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http://dx.doi.org/10.1016/j.cell.2019.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6625921PMC
July 2019

The purine biosynthesis regulator PurR moonlights as a virulence regulator in .

Proc Natl Acad Sci U S A 2019 07 19;116(27):13563-13572. Epub 2019 Jun 19.

Department of Microbiology, New York University School of Medicine, New York, NY 10016;

The pathogen colonizes and infects a variety of different sites within the human body. To adapt to these different environments, relies on a complex and finely tuned regulatory network. While some of these networks have been well-elucidated, the functions of more than 50% of the transcriptional regulators in remain unexplored. Here, we assess the contribution of the LacI family of metabolic regulators to staphylococcal virulence. We found that inactivating the purine biosynthesis regulator resulted in a strain that was acutely virulent in bloodstream infection models in mice and in ex vivo models using primary human neutrophils. Remarkably, these enhanced pathogenic traits are independent of purine biosynthesis, as the mutant was still highly virulent in the presence of mutations that disrupt PurR's canonical role. Through the use of transcriptomics coupled with proteomics, we revealed that a number of virulence factors are differentially regulated in the absence of Indeed, we demonstrate that PurR directly binds to the promoters of genes encoding virulence factors and to master regulators of virulence. These results guided us into further ex vivo and in vivo studies, where we discovered that toxins drive the death of human phagocytes and mice, whereas the surface adhesin FnbA contributes to the increased bacterial burden observed in the mutant. Thus, repurposes a metabolic regulator to directly control the expression of virulence factors, and by doing so, tempers its pathogenesis.
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http://dx.doi.org/10.1073/pnas.1904280116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6613142PMC
July 2019

Affinity purification mass spectrometry analysis of PD-1 uncovers SAP as a new checkpoint inhibitor.

Proc Natl Acad Sci U S A 2018 01 27;115(3):E468-E477. Epub 2017 Dec 27.

Department of Medicine, New York University School of Medicine, New York, NY 10016;

Programmed cell death-1 (PD-1) is an essential inhibitory receptor in T cells. Antibodies targeting PD-1 elicit durable clinical responses in patients with multiple tumor indications. Nevertheless, a significant proportion of patients do not respond to anti-PD-1 treatment, and a better understanding of the signaling pathways downstream of PD-1 could provide biomarkers for those whose tumors respond and new therapeutic approaches for those whose tumors do not. We used affinity purification mass spectrometry to uncover multiple proteins associated with PD-1. Among these proteins, signaling lymphocytic activation molecule-associated protein (SAP) was functionally and mechanistically analyzed for its contribution to PD-1 inhibitory responses. Silencing of SAP augmented and overexpression blocked PD-1 function. T cells from patients with X-linked lymphoproliferative disease (XLP), who lack functional SAP, were hyperresponsive to PD-1 signaling, confirming its inhibitory role downstream of PD-1. Strikingly, signaling downstream of PD-1 in purified T cell subsets did not correlate with PD-1 surface expression but was inversely correlated with intracellular SAP levels. Mechanistically, SAP opposed PD-1 function by acting as a molecular shield of key tyrosine residues that are targets for the tyrosine phosphatase SHP2, which mediates PD-1 inhibitory properties. Our results identify SAP as an inhibitor of PD-1 function and SHP2 as a potential therapeutic target in patients with XLP.
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http://dx.doi.org/10.1073/pnas.1710437115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5776966PMC
January 2018

Proteomic differences in amyloid plaques in rapidly progressive and sporadic Alzheimer's disease.

Acta Neuropathol 2017 06 4;133(6):933-954. Epub 2017 Mar 4.

Department of Neurology, Center for Cognitive Neurology, NYU School of Medicine, New York, NY, USA.

Rapidly progressive Alzheimer's disease (rpAD) is a particularly aggressive form of Alzheimer's disease, with a median survival time of 7-10 months after diagnosis. Why these patients have such a rapid progression of Alzheimer's disease is currently unknown. To further understand pathological differences between rpAD and typical sporadic Alzheimer's disease (sAD) we used localized proteomics to analyze the protein differences in amyloid plaques in rpAD and sAD. Label-free quantitative LC-MS/MS was performed on amyloid plaques microdissected from rpAD and sAD patients (n = 22 for each patient group) and protein expression differences were quantified. On average, 913 ± 30 (mean ± SEM) proteins were quantified in plaques from each patient and 279 of these proteins were consistently found in plaques from every patient. We found significant differences in protein composition between rpAD and sAD plaques. We found that rpAD plaques contained significantly higher levels of neuronal proteins (p = 0.0017) and significantly lower levels of astrocytic proteins (p = 1.08 × 10). Unexpectedly, cumulative protein differences in rpAD plaques did not suggest accelerated typical sAD. Plaques from patients with rpAD were particularly abundant in synaptic proteins, especially those involved in synaptic vesicle release, highlighting the potential importance of synaptic dysfunction in the accelerated development of plaque pathology in rpAD. Combined, our data provide new direct evidence that amyloid plaques do not all have the same protein composition and that the proteomic differences in plaques could provide important insight into the factors that contribute to plaque development. The cumulative protein differences in rpAD plaques suggest rpAD may be a novel subtype of Alzheimer's disease.
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http://dx.doi.org/10.1007/s00401-017-1691-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5503748PMC
June 2017

Using Quantitative Spectrometry to Understand the Influence of Genetics and Nutritional Perturbations On the Virulence Potential of .

Mol Cell Proteomics 2017 04 14;16(4 suppl 1):S15-S28. Epub 2017 Feb 14.

From the ‡Proteomics Laboratory,

() is the leading cause of a variety of bacterial infections ranging from superficial skin infections to invasive and life threatening diseases such as septic bacteremia, necrotizing pneumonia, and endocarditis. The success of as a human pathogen is contributed to its ability to adapt to different environments by changing expression, production, or secretion of virulence factors. Although immune evasion is well-studied, the regulation of virulence factors under different nutrient and growth conditions is still not well understood. Here, we used label-free quantitative mass spectrometry to quantify and compare the exoproteins ( exoproteomes) of master regulator mutants or established reference strains. Different environmental conditions were addressed by growing the bacteria in rich or minimal media at different phases of growth. We observed clear differences in the composition of the exoproteomes depending on the genetic background or growth conditions. The relative abundance of cytotoxins determined in our study correlated well with differences in cytotoxicity measured by lysis of human neutrophils. Our findings demonstrate that label-free quantitative mass spectrometry is a versatile tool for predicting the virulence of bacterial strains and highlights the importance of the experimental design for studies. Furthermore, the results indicate that label-free proteomics can be used to cluster isolates into groups with similar virulence properties, highlighting the power of label-free quantitative mass spectrometry to distinguish strains.
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http://dx.doi.org/10.1074/mcp.O116.065581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5393389PMC
April 2017

The arc of Mass Spectrometry Exchange Formats is long, but it bends toward HDF5.

Mass Spectrom Rev 2017 09 14;36(5):668-673. Epub 2016 Oct 14.

Research Division, Weill Cornell Medicine in Qatar, Doha, State of Qatar.

The evolution of data exchange in Mass Spectrometry spans decades and has ranged from human-readable text files representing individual scans or collections thereof (McDonald et al., 2004) through the official standard XML-based (Harold, Means, & Udemadu, 2005) data interchange standard (Deutsch, 2012), to increasingly compressed (Teleman et al., 2014) variants of this standard sometimes requiring purely binary adjunct files (Römpp et al., 2011). While the desire to maintain even partial human readability is understandable, the inherent mismatch between XML's textual and irregular format relative to the numeric and highly regular nature of actual spectral data, along with the explosive growth in dataset scales and the resulting need for efficient (binary and indexed) access has led to a phenomenon referred to as "technical drift" (Davis, 2013). While the drift is being continuously corrected using adjunct formats, compression schemes, and programs (Röst et al., 2015), we propose that the future of Mass Spectrometry Exchange Formats lies in the continued reliance and development of the PSI-MS (Mayer et al., 2014) controlled vocabulary, along with an expedited shift to an alternative, thriving and well-supported ecosystem for scientific data-exchange, storage, and access in binary form, namely that of HDF5 (Koranne, 2011). Indeed, pioneering efforts to leverage this universal, binary, and hierarchical data-format have already been published (Wilhelm et al., 2012; Rübel et al., 2013) though they have under-utilized self-description, a key property shared by HDF5 and XML. We demonstrate that a straightforward usage of plain ("vanilla") HDF5 yields immediate returns including, but not limited to, highly efficient data access, platform independent data viewers, a variety of libraries (Collette, 2014) for data retrieval and manipulation in many programming languages and remote data access through comprehensive RESTful data-servers. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:668-673, 2017.
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http://dx.doi.org/10.1002/mas.21522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6088231PMC
September 2017

OpenSlice: Quantitative data sharing from HyperPeaks to global ion chromatograms (GICs).

Proteomics 2016 09;16(18):2495-501

Institute for Systems Genetics and Department for Biochemistry and Molecular Pharmacology, NYU School of Medicine, New York, NY, USA.

Data sharing in the field of MS has advanced greatly thanks to innovations such as the standardized formats, data repositories, and publications guidelines. However, there is currently no data sharing mechanism that enables real-time data browsing and deep linking on a large scale: unrestricted data access (particularly at the quantitative level) ultimately requires the user to download a local copy of the relevant data files (e.g., in order to generate extracted ion chromatograms [XICs]). In this technical resource, we present a set of technologies (collectively termed OpenSlice) that enable the user to quantitatively query hundreds of hours of proteomics discovery data (i.e., nontargeted acquisition) in real time: the user is able to effectively generate XICs for arbitrary masses on the fly and across the entire dataset (so-called global ion chromatograms), interacting with the results through a very intuitive browser-based interface. A key design consideration underlying the OpenSlice approach is the notion that every aspect of the acquired data must be accessible through a RESTful uniform resource locator based application programming interface, up to and including individual chromatographic peaks (hence HyperPeaks). A publicly accessible demonstration of this technology based on the Clinical Proteomics Tumor Analysis Consortium CompRef dataset is made available at http://compref.fenyolab.org.
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http://dx.doi.org/10.1002/pmic.201600096DOI Listing
September 2016

PGx: Putting Peptides to BED.

J Proteome Res 2016 Mar 18;15(3):795-9. Epub 2015 Dec 18.

NYU Langone Medical Center , 227 East 30th Street, New York, New York 10016, United States.

Every molecular player in the cast of biology's central dogma is being sequenced and quantified with increasing ease and coverage. To bring the resulting genomic, transcriptomic, and proteomic data sets into coherence, tools must be developed that do not constrain data acquisition and analytics in any way but rather provide simple links across previously acquired data sets with minimal preprocessing and hassle. Here we present such a tool: PGx, which supports proteogenomic integration of mass spectrometry proteomics data with next-generation sequencing by mapping identified peptides onto their putative genomic coordinates.
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http://dx.doi.org/10.1021/acs.jproteome.5b00870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4782174PMC
March 2016

An Analysis of the Sensitivity of Proteogenomic Mapping of Somatic Mutations and Novel Splicing Events in Cancer.

Mol Cell Proteomics 2016 Mar 2;15(3):1060-71. Epub 2015 Dec 2.

¶Washington University in St. Louis, St. Louis, MO;

Improvements in mass spectrometry (MS)-based peptide sequencing provide a new opportunity to determine whether polymorphisms, mutations, and splice variants identified in cancer cells are translated. Herein, we apply a proteogenomic data integration tool (QUILTS) to illustrate protein variant discovery using whole genome, whole transcriptome, and global proteome datasets generated from a pair of luminal and basal-like breast-cancer-patient-derived xenografts (PDX). The sensitivity of proteogenomic analysis for singe nucleotide variant (SNV) expression and novel splice junction (NSJ) detection was probed using multiple MS/MS sample process replicates defined here as an independent tandem MS experiment using identical sample material. Despite analysis of over 30 sample process replicates, only about 10% of SNVs (somatic and germline) detected by both DNA and RNA sequencing were observed as peptides. An even smaller proportion of peptides corresponding to NSJ observed by RNA sequencing were detected (<0.1%). Peptides mapping to DNA-detected SNVs without a detectable mRNA transcript were also observed, suggesting that transcriptome coverage was incomplete (∼80%). In contrast to germline variants, somatic variants were less likely to be detected at the peptide level in the basal-like tumor than in the luminal tumor, raising the possibility of differential translation or protein degradation effects. In conclusion, this large-scale proteogenomic integration allowed us to determine the degree to which mutations are translated and identify gaps in sequence coverage, thereby benchmarking current technology and progress toward whole cancer proteome and transcriptome analysis.
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http://dx.doi.org/10.1074/mcp.M115.056226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4813688PMC
March 2016

Protected amine labels: a versatile molecular scaffold for multiplexed nominal mass and sub-Da isotopologue quantitative proteomic reagents.

J Am Soc Mass Spectrom 2014 Apr 5;25(4):636-50. Epub 2014 Feb 5.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, 02215-5450, USA.

We assemble a versatile molecular scaffold from simple building blocks to create binary and multiplexed stable isotope reagents for quantitative mass spectrometry. Termed Protected Amine Labels (PAL), these reagents offer multiple analytical figures of merit including, (1) robust targeting of peptide N-termini and lysyl side chains, (2) optimal mass spectrometry ionization efficiency through regeneration of primary amines on labeled peptides, (3) an amino acid-based mass tag that incorporates heavy isotopes of carbon, nitrogen, and oxygen to ensure matched physicochemical and MS/MS fragmentation behavior among labeled peptides, and (4) a molecularly efficient architecture, in which the majority of hetero-atom centers can be used to synthesize a variety of nominal mass and sub-Da isotopologue stable isotope reagents. We demonstrate the performance of these reagents in well-established strategies whereby up to four channels of peptide isotopomers, each separated by 4 Da, are quantified in MS-level scans with accuracies comparable to current commercial reagents. In addition, we utilize the PAL scaffold to create isotopologue reagents in which labeled peptide analogs differ in mass based on the binding energy in carbon and nitrogen nuclei, thereby allowing quantification based on MS or MS/MS spectra. We demonstrate accurate quantification for reagents that support 6-plex labeling and propose extension of this scheme to 9-channels based on a similar PAL scaffold. Finally, we provide exemplar data that extend the application of isotopologe-based quantification reagents to medium resolution, quadrupole time-of-flight mass spectrometers.
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http://dx.doi.org/10.1007/s13361-013-0811-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3971929PMC
April 2014

Library dependent LC-MS/MS acquisition via mzAPI/Live.

Proteomics 2013 May;13(9):1412-6

Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA 02215-5450, USA.

The use of MS for characterization of small molecules, nucleotides, and proteins in model organisms as well as primary tissues and clinical samples continues to proliferate at a rapid pace. The complexity and dynamic range of target analytes in biological systems hinders comprehensive analysis and simultaneously drives improvements in instrument hardware and software. As a result, state-of-the-art commercial mass spectrometers are equipped with sophisticated embedded control systems that provide robust acquisition methods accessed through intuitive graphical interfaces. Although optimized for speed, these preconfigured scan functions are otherwise closed to end-user customization beyond simple, analytical-centric parameters supplied by the manufacturer. Here, we present an open-source framework (mzAPI/Live) that enables users to generate arbitrarily complex LC-MS(n) acquisition methods via simple Python scripting. As a powerful proof-of-concept, we demonstrate real-time assignment of tandem mass spectra through rapid query of NIST peptide libraries. This represents an unprecedented capability to make acquisition decisions based on knowledge of analyte structures determined during the run itself, thus providing a path toward biology-driven MS data acquisition for the broader community.
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http://dx.doi.org/10.1002/pmic.201200583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3779148PMC
May 2013

Short toxin-like proteins abound in Cnidaria genomes.

Toxins (Basel) 2012 Nov 16;4(11):1367-84. Epub 2012 Nov 16.

Department of Biological Chemistry, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.

Cnidaria is a rich phylum that includes thousands of marine species. In this study, we focused on Anthozoa and Hydrozoa that are represented by the Nematostella vectensis (Sea anemone) and Hydra magnipapillata genomes. We present a method for ranking the toxin-like candidates from complete proteomes of Cnidaria. Toxin-like functions were revealed using ClanTox, a statistical machine-learning predictor trained on ion channel inhibitors from venomous animals. Fundamental features that were emphasized in training ClanTox include cysteines and their spacing along the sequences. Among the 83,000 proteins derived from Cnidaria representatives, we found 170 candidates that fulfill the properties of toxin-like-proteins, the vast majority of which were previously unrecognized as toxins. An additional 394 short proteins exhibit characteristics of toxin-like proteins at a moderate degree of confidence. Remarkably, only 11% of the predicted toxin-like proteins were previously classified as toxins. Based on our prediction methodology and manual annotation, we inferred functions for over 400 of these proteins. Such functions include protease inhibitors, membrane pore formation, ion channel blockers and metal binding proteins. Many of the proteins belong to small families of paralogs. We conclude that the evolutionary expansion of toxin-like proteins in Cnidaria contributes to their fitness in the complex environment of the aquatic ecosystem.
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http://dx.doi.org/10.3390/toxins4111367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3509713PMC
November 2012

Implicit biology in peptide spectral libraries.

Anal Chem 2012 Sep 31;84(18):7919-25. Epub 2012 Aug 31.

Department of Biological Chemistry, Hebrew University of Jerusalem, Israel.

Mass spectral libraries are collections of mass spectra curated specifically to facilitate the identification of small molecules, metabolites, and short peptides. One of the most comprehensive peptide spectral libraries is curated by NIST and contains upward of half a million annotated spectra dominated by human and model organisms including budding yeast and mouse. While motivated primarily by the technological goal of increasing sensitivity and specificity in spectral identification, we have found that the NIST spectral library constitutes a surprisingly rich source of biological knowledge. In this Article, we show that data-mining of these published libraries while applying strict empirical thresholds yields many characteristics of protein biology. In particular, we demonstrate that the size and increasingly comprehensive nature of these libraries, generated from whole-proteome digests, enables inference from the presence but crucially also from the absence of spectra for individual peptides. We illustrate implicit biological trends that lead to significant absence of spectra accounted for by complex post-translational modifications and overlooked proteolytic sites. We conclude that many subtle biological signatures such as genetic variants, regulated proteolysis, and post-translational modifications are exposed through the systematic mining of spectral collections originally compiled as general-purpose, technology-oriented resources.
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http://dx.doi.org/10.1021/ac301674yDOI Listing
September 2012

Proteomic analysis demonstrates activator- and chromatin-specific recruitment to promoters.

J Biol Chem 2012 Oct 17;287(42):35397-35408. Epub 2012 Aug 17.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115; Department of Cancer Biology, Harvard Medical School, Boston, Massachusetts 02115; Blais Proteomics Center, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115. Electronic address:

In-depth characterization of RNA polymerase II preinitiation complexes remains an important and challenging goal. We used quantitative mass spectrometry to explore context-dependent Saccharomyces cerevisiae preinitiation complex formation at the HIS4 promoter reconstituted on naked and chromatinized DNA templates. The transcription activators Gal4-VP16 and Gal4-Gcn4 recruited a limited set of chromatin-related coactivator complexes, namely the chromatin remodeler Swi/Snf and histone acetyltransferases SAGA and NuA4, suggesting that transcription stimulation is mediated through these factors. Moreover, the two activators differentially recruited the coactivator complexes, consistent with specific activator-coactivator interactions. Chromatinized templates suppressed recruitment of basal transcription factors, thereby amplifying the effect of activators, compared with naked DNA templates. This system is sensitive, highly reproducible, and easily applicable to mapping the repertoire of proteins found at any promoter.
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http://dx.doi.org/10.1074/jbc.M112.391581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3471693PMC
October 2012

Interpreting cancer genomes using systematic host network perturbations by tumour virus proteins.

Nature 2012 Jul;487(7408):491-5

Genomic Analysis of Network Perturbations Center of Excellence in Genomic Science, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA.

Genotypic differences greatly influence susceptibility and resistance to disease. Understanding genotype-phenotype relationships requires that phenotypes be viewed as manifestations of network properties, rather than simply as the result of individual genomic variations. Genome sequencing efforts have identified numerous germline mutations, and large numbers of somatic genomic alterations, associated with a predisposition to cancer. However, it remains difficult to distinguish background, or 'passenger', cancer mutations from causal, or 'driver', mutations in these data sets. Human viruses intrinsically depend on their host cell during the course of infection and can elicit pathological phenotypes similar to those arising from mutations. Here we test the hypothesis that genomic variations and tumour viruses may cause cancer through related mechanisms, by systematically examining host interactome and transcriptome network perturbations caused by DNA tumour virus proteins. The resulting integrated viral perturbation data reflects rewiring of the host cell networks, and highlights pathways, such as Notch signalling and apoptosis, that go awry in cancer. We show that systematic analyses of host targets of viral proteins can identify cancer genes with a success rate on a par with their identification through functional genomics and large-scale cataloguing of tumour mutations. Together, these complementary approaches increase the specificity of cancer gene identification. Combining systems-level studies of pathogen-encoded gene products with genomic approaches will facilitate the prioritization of cancer-causing driver genes to advance the understanding of the genetic basis of human cancer.
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http://dx.doi.org/10.1038/nature11288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3408847PMC
July 2012

Quantitative assessment of whole-body tumor burden in adult patients with neurofibromatosis.

PLoS One 2012 27;7(4):e35711. Epub 2012 Apr 27.

Department of Neurology and Cancer Center, Massachusetts General Hospital, Boston, Massachusetts, United States of America.

Purpose: Patients with neurofibromatosis 1 (NF1), NF2, and schwannomatosis are at risk for multiple nerve sheath tumors and premature mortality. Traditional magnetic resonance imaging (MRI) has limited ability to assess disease burden accurately. The aim of this study was to establish an international cohort of patients with quantified whole-body internal tumor burden and to correlate tumor burden with clinical features of disease.

Methods: We determined the number, volume, and distribution of internal nerve sheath tumors in patients using whole-body MRI (WBMRI) and three-dimensional computerized volumetry. We quantified the distribution of tumor volume across body regions and used unsupervised cluster analysis to group patients based on tumor distribution. We correlated the presence and volume of internal tumors with disease-related and demographic factors.

Results: WBMRI identified 1286 tumors in 145/247 patients (59%). Schwannomatosis patients had the highest prevalence of tumors (P = 0.03), but NF1 patients had the highest median tumor volume (P = 0.02). Tumor volume was unevenly distributed across body regions with overrepresentation of the head/neck and pelvis. Risk factors for internal nerve sheath tumors included decreasing numbers of café-au-lait macules in NF1 patients (P = 0.003) and history of skeletal abnormalities in NF2 patients (P = 0.09). Risk factors for higher tumor volume included female gender (P = 0.05) and increasing subcutaneous neurofibromas (P = 0.03) in NF1 patients, absence of cutaneous schwannomas in NF2 patients (P = 0.06), and increasing age in schwannomatosis patients (p = 0.10).

Conclusion: WBMRI provides a comprehensive phenotype of neurofibromatosis patients, identifies distinct anatomic subgroups, and provides the basis for investigating molecular biomarkers that correlate with unique disease manifestations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035711PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338705PMC
September 2012

DNA ends alter the molecular composition and localization of Ku multicomponent complexes.

Mol Cell Proteomics 2012 Aug 24;11(8):411-21. Epub 2012 Apr 24.

Department of Cancer Biology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, Massachusetts, 02215-5450, USA.

The Ku heterodimer plays an essential role in non-homologous end-joining and other cellular processes including transcription, telomere maintenance and apoptosis. While the function of Ku is regulated through its association with other proteins and nucleic acids, the specific composition of these macromolecular complexes and their dynamic response to endogenous and exogenous cellular stimuli are not well understood. Here we use quantitative proteomics to define the composition of Ku multicomponent complexes and demonstrate that they are dramatically altered in response to UV radiation. Subsequent biochemical assays revealed that the presence of DNA ends leads to the substitution of RNA-binding proteins with DNA and chromatin associated factors to create a macromolecular complex poised for DNA repair. We observed that dynamic remodeling of the Ku complex coincided with exit of Ku and other DNA repair proteins from the nucleolus. Microinjection of sheared DNA into live cells as a mimetic for double strand breaks confirmed these findings in vivo.
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http://dx.doi.org/10.1074/mcp.M111.013581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3412971PMC
August 2012

Delayed fragmentation and optimized isolation width settings for improvement of protein identification and accuracy of isobaric mass tag quantification on Orbitrap-type mass spectrometers.

Anal Chem 2011 Dec 4;83(23):8959-67. Epub 2011 Nov 4.

Cellzome AG, Meyerhofstrasse 1, 69117 Heidelberg, Germany.

Fragmentation of multiple peptides in a single tandem mass scan impairs accuracy of isobaric mass tag based quantification. Consequently, practitioners aim at fragmenting peptide ions with the highest possible purity without compromising on sensitivity and coverage achieved in the experiment. Here we report the first systematic study optimizing delayed fragmentation options on Orbitrap instruments. We demonstrate that by delaying peptide fragmentation to occur closer to the apex of the chromatographic peak in liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiments cofragmentation is reduced by 2-fold and peptides are fragmented with 2.8-fold better signal-to-noise ratios. This results in significantly improved accuracy of isobaric mass tag quantification. Further, we measured cofragmentation dependence on isolation width. In comparison to Orbitrap XL instruments the reduced space charging in the Orbitrap Velos enables isolation widths as narrow as 1 Th without impairing coverage, thus substantially reducing cofragmentation. When delayed peptide fragmentation and narrow isolation width settings were both applied, cofragmentation-induced ratio compression could be reduced by 32% on a log2 scale under otherwise identical conditions.
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http://dx.doi.org/10.1021/ac201760xDOI Listing
December 2011

ARISTO: ontological classification of small molecules by electron ionization-mass spectrometry.

Nucleic Acids Res 2011 Jul 27;39(Web Server issue):W505-10. Epub 2011 May 27.

Sudarsky Center for Computational Biology, Department of Biological Chemistry, Hebrew University of Jerusalem, Israel.

Gas chromatography-mass spectrometry (GC-MS) acquisitions routinely yield hundreds to thousands of Electron Ionization (EI) mass spectra. The chemical identification of these spectra typically involves a search protocol that seeks an exact match to a reference spectrum. Reference spectra are found in comprehensive libraries of small molecule EI spectra curated by commercial and public entities. We developed ARISTO (Automatic Reduction of Ion Spectra To Ontology), a webtool, which provides information regarding the general chemical nature of the compound underlying an input EI mass spectrum. Importantly, ARISTO can provide such annotation without necessitating an exact match to a specific compound. ARISTO provides assignments to a subset of the ChEBI (Chemical Entities of Biological Interest) dictionary, an ontology, which aims to cover biologically relevant small molecules. Our system takes as input a mass spectrum represented as a series of mass and intensity pairs; the system returns a graphical representation of the supported ontology as well as a detailed table of suggested annotations along with their associated statistical evidence. ARISTO is accessible at this URL: http://www.ionspectra.org/aristo. The system is free, open to all and does not require registration of any sort.
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http://dx.doi.org/10.1093/nar/gkr403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3125788PMC
July 2011

mzServer: web-based programmatic access for mass spectrometry data analysis.

Mol Cell Proteomics 2011 May 25;10(5):M110.003988. Epub 2011 Jan 25.

Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, Harvard Medical School,Boston, MA 02115, USA.

Continued progress toward systematic generation of large-scale and comprehensive proteomics data in the context of biomedical research will create project-level data sets of unprecedented size and ultimately overwhelm current practices for results validation that are based on distribution of native or surrogate mass spectrometry files. Moreover, the majority of proteomics studies leverage discovery-mode MS/MS analyses, rendering associated data-reduction efforts incomplete at best, and essentially ensuring future demand for re-analysis of data as new biological and technical information become available. Based on these observations, we propose to move beyond the sharing of interpreted spectra, or even the distribution of data at the individual file or project level, to a system much like that used in high-energy physics and astronomy, whereby raw data are made programmatically accessible at the site of acquisition. Toward this end we have developed a web-based server (mzServer), which exposes our common API (mzAPI) through very intuitive (RESTful) uniform resource locators (URL) and provides remote data access and analysis capabilities to the research community. Our prototype mzServer provides a model for lab-based and community-wide data access and analysis.
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http://dx.doi.org/10.1074/mcp.M110.003988DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3098585PMC
May 2011

mzResults: an interactive viewer for interrogation and distribution of proteomics results.

Mol Cell Proteomics 2011 May 25;10(5):M110.003970. Epub 2011 Jan 25.

Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute Boston, MA 02115-6084, USA

The growing use of mass spectrometry in the context of biomedical research has been accompanied by an increased demand for distribution of results in a format that facilitates rapid and efficient validation of claims by reviewers and other interested parties. However, the continued evolution of mass spectrometry hardware, sample preparation methods, and peptide identification algorithms complicates standardization and creates hurdles related to compliance with journal submission requirements. Moreover, the recently announced Philadelphia Guidelines (1, 2) suggest that authors provide native mass spectrometry data files in support of their peer-reviewed research articles. These trends highlight the need for data viewers and other tools that work independently of manufacturers' proprietary data systems and seamlessly connect proteomics results with original data files to support user-driven data validation and review. Based upon our recently described API(1)-based framework for mass spectrometry data analysis (3, 4), we created an interactive viewer (mzResults) that is built on established database standards and enables efficient distribution and interrogation of results associated with proteomics experiments, while also providing a convenient mechanism for authors to comply with data submission standards as described in the Philadelphia Guidelines. In addition, the architecture of mzResults supports in-depth queries of the native mass spectrometry files through our multiplierz software environment. We use phosphoproteomics data to illustrate the features and capabilities of mzResults.
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http://dx.doi.org/10.1074/mcp.M110.003970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3098584PMC
May 2011

The complete peptide dictionary--a meta-proteomics resource.

Proteomics 2010 Dec;10(23):4306-10

Department of Biological Chemistry, The Hebrew University of Jerusalem, Israel.

Recent developments in MS-based proteomics have increased the emphasis on peptides as a primary observable. While peptides are identified by tandem mass spectra, the link between peptide and protein remains implicit given the bottom-up nature of the experiment in which proteins are enzymatically digested prior to sequencing. It is therefore useful to provide a fast lookup from peptide to protein in order to systematically establish the broadest possible protein basis for the observed peptides. Here, we describe Pep2Pro, a fast web-service providing protein lookup by peptides covering the entire protein space comprising ∼10 million UniRef100 sequences. We demonstrate the usefulness of the service by reanalyzing peptides from two recent meta-proteomic data sets and identifying taxon-specific peptides, thereby implicating individual species as being present in these complex samples. The Pep2Pro web service can be accessed at http://www.pep2pro.org.
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http://dx.doi.org/10.1002/pmic.201000270DOI Listing
December 2010

A QUICK screen for Lrrk2 interaction partners--leucine-rich repeat kinase 2 is involved in actin cytoskeleton dynamics.

Mol Cell Proteomics 2011 Jan 27;10(1):M110.001172. Epub 2010 Sep 27.

Department of Protein Science, Helmholtz Zentrum München-German Research Center for Environmental Health (GmbH), 85764 Neuherberg, Germany.

Mutations in human leucine-rich repeat kinase 2 (Lrrk2), a protein of yet unknown function, are linked to Parkinson's disease caused by degeneration of midbrain dopaminergic neurons. The protein comprises several domains including a GTPase and a kinase domain both affected by several pathogenic mutations. To elucidate the molecular interaction network of endogenous Lrrk2 under stoichiometric constraints, we applied QUICK (quantitative immunoprecipitation combined with knockdown) in NIH3T3 cells. The identified interactome reveals actin isoforms as well as actin-associated proteins involved in actin filament assembly, organization, rearrangement, and maintenance, suggesting that the biological function of Lrrk2 is linked to cytoskeletal dynamics. In fact, we demonstrate Lrrk2 de novo binding to F-actin and its ability to modulate its assembly in vitro. When tested in intact cells, knockdown of Lrrk2 causes morphological alterations in NIH3T3 cells. In developing dopaminergic midbrain primary neurons, Lrrk2 knockdown results in shortened neurite processes, indicating a physiological role of Lrrk2 in cytoskeletal organization and dynamics of dopaminergic neurons. Hence, our results demonstrate that molecular interactions as well as the physiological function of Lrrk2 are closely related to the organization of the actin-based cytoskeleton, a crucial feature of neuronal development and neuron function.
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http://dx.doi.org/10.1074/mcp.M110.001172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3013447PMC
January 2011

A predictor for toxin-like proteins exposes cell modulator candidates within viral genomes.

Bioinformatics 2010 Sep;26(18):i482-8

School of Computer Science and Engineering, Hebrew University of Jerusalem, Israel.

Motivation: Animal toxins operate by binding to receptors and ion channels. These proteins are short and vary in sequence, structure and function. Sporadic discoveries have also revealed endogenous toxin-like proteins in non-venomous organisms. Viral proteins are the largest group of quickly evolving proteomes. We tested the hypothesis that toxin-like proteins exist in viruses and that they act to modulate functions of their hosts.

Results: We updated and improved a classifier for compact proteins resembling short animal toxins that is based on a machine-learning method. We applied it in a large-scale setting to identify toxin-like proteins among short viral proteins. Among the approximately 26 000 representatives of such short proteins, 510 sequences were positively identified. We focused on the 19 highest scoring proteins. Among them, we identified conotoxin-like proteins, growth factors receptor-like proteins and anti-bacterial peptides. Our predictor was shown to enhance annotation inference for many 'uncharacterized' proteins. We conclude that our protocol can expose toxin-like proteins in unexplored niches including metagenomics data and enhance the systematic discovery of novel cell modulators for drug development.

Availability: ClanTox is available at http://www.clantox.cs.huji.ac.il.
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http://dx.doi.org/10.1093/bioinformatics/btq375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2935411PMC
September 2010