DOCSLIB.ORG

DIA-Based Systems Biology Approach Unveils E3 Ubiquitin Ligase-Dependent Responses to a Metabolic Shift

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

DIA-based systems biology approach unveils E3 ubiquitin ligase-dependent responses to a metabolic shift Ozge Karayela,1, André C. Michaelisa, Matthias Manna,2, Brenda A. Schulmanb,2, and Christine R. Langloisb,1,2 aDepartment of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany; and bDepartment of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany Contributed by Brenda A. Schulman, November 8, 2020 (sent for review September 28, 2020; reviewed by Angus I. Lamond and Alexander Varshavsky) The yeast Saccharomyces cerevisiae is a powerful model system (9), and the recently developed SWAp-tag library (10–12), has for systems-wide biology screens and large-scale proteomics meth- made yeast a premier model system for conducting transcriptomics, ods. Nearly complete proteomics coverage has been achieved owing proteomics, interactomics, or metabolomics screens (13–19). In- to advances in mass spectrometry. However, it remains challenging to deed, systems-wide biology screens and large-scale proteomics were scale this technology for rapid and high-throughput analysis of the both pioneered in the yeast model. Furthermore, the cellular in- yeast proteome to investigate biological pathways on a global scale. teraction networks and molecular mechanisms ascertained in yeast Here we describe a systems biology workflow employing plate-based can be readily applied to other systems (20–22). sample preparation and rapid, single-run, data-independent mass Early genome-wide studies showed that over 4,000 proteins are spectrometry analysis (DIA). Our approach is straightforward, easy expressed during log-phase growth in yeast and this organism was to implement, and enables quantitative profiling and comparisons the first whose entire proteome was mapped by mass spectrometry of hundreds of nearly complete yeast proteomes in only a few days. (MS)-based proteomics (23). Subsequently, yeast has served as a We evaluate its capability by characterizing changes in the yeast pro- model of choice for the development of ever-more-sensitive and teome in response to environmental perturbations, identifying dis- faster proteomics workflows (23–34). Remarkably, the optimized tinct responses to each of them and providing a comprehensive sample preparation coupled with MS analysis performed on the resource of these responses. Apart from rapidly recapitulating previ- Orbitrap hybrid mass spectrometer allowed identification of SYSTEMS BIOLOGY ously observed responses, we characterized carbon source-dependent around 4,000 yeast proteins over a 70-min liquid chromatography regulation of the GID E3 ligase, an important regulator of cellular (LC)-MS/MS run (24, 30). However, the necessity of technological metabolism during the switch between gluconeogenic and glycolytic expertise and lengthy analysis times for high-quality, in-depth growth conditions. This unveiled regulatory targets of the GID ligase yeast proteome measurements has so far precluded the wide- during a metabolic switch. Our comprehensive yeast system readout spread adoption of cutting-edge proteomics workflows in the yeast pinpointed effects of a single deletion or point mutation in the GID research community. With further advances in technology and complex on the global proteome, allowing the identification and val- new acquisition modes, such as data-independent acquisition idation of targets of the GID E3 ligase. Moreover, this approach (DIA) (35, 36), we hypothesized that it would now be possible to allowed the identification of targets from multiple cellular pathways obtain accurate and high yeast proteome coverage by a straight- that display distinct patterns of regulation. Although developed in forward and rapid single-run approach, enabling researchers to yeast, rapid whole-proteome–based readouts can serve as compre- hensive systems-level assays in all cellular systems. Significance yeast systems biology | mass spectrometry | proteomics | stress | GID E3 ligase We use a single-run, data-independent acquisition–based mass spectrometry approach to generate and compare dozens of yeast proteomes in less than a day, and provide a compre- roteome remodeling has repeatedly proven to be a vital hensive resource detailing changes to the yeast proteome fol- cellular mechanism in response to stress, changes in envi- P lowing commonly used stress treatments in yeast. Our systems ronmental conditions, and toxins or pathogens. Cells must both biology approach identifies and validates regulatory targets of synthesize proteins which enable them to adapt to the new envi- an E3 ubiquitin ligase during a metabolic switch, providing ronmental condition and inactivate or degrade proteins which are insights into the interplay of metabolic pathways. The speed, detrimental or no longer needed. For each environmental simplicity, and scalability of this workflow makes it particularly perturbation, the proteome must be precisely and distinctly well-suited for screens in any cellular system to investigate remodeled to ensure healthy and viable cells (1). Indeed, de- specific effects of deletions or mutants or other perturbations creases in proteome integrity are hallmarks of many human to obtain the response of biological system on a global level. diseases, including cancer, Alzheimer’s disease, muscular dys- – trophies, and cystic fibrosis (2 4). Despite the importance of Author contributions: O.K., M.M., B.A.S., and C.R.L. designed research; O.K. and C.R.L. cellular stress responses, our understanding of how cellular performed research; O.K., A.C.M., and C.R.L. contributed new reagents/analytic tools; O.K. pathways interact during adaptation remains incomplete. and C.R.L. analyzed data; and O.K., M.M., B.A.S., and C.R.L. wrote the paper. Therefore, knowing precisely how the proteome changes at a Reviewers: A.I.L., University of Dundee; and A.V., California Institute of Technology. global level in response to environmental cues is crucial for The authors declare no competing interest. identifying the underlying molecular mechanisms that facilitate This open access article is distributed under Creative Commons Attribution-NonCommercial- cellular adaptation. NoDerivatives License 4.0 (CC BY-NC-ND). The yeast Saccharomyces cerevisiae is a powerful model system 1O.K. and C.R.L. contributed equally to this work. that is widely used to probe biological pathways, due to its ease 2To whom correspondence may be addressed. Email: [email protected], of manipulation and rapid growth compared to mammalian [email protected], or [email protected]. models. In addition, the availability of extensive genetic re- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ sources in yeast, including deletion libraries (5, 6), green fluo- doi:10.1073/pnas.2020197117/-/DCSupplemental. rescent protein–tagged libraries (7, 8), overexpression libraries www.pnas.org/cgi/doi/10.1073/pnas.2020197117 PNAS Latest Articles | 1of10 Downloaded by guest on September 27, 2021 easily study biological processes on a global scale. Such a system Combined with our own comprehensive spectral library, the could then serve as a template for more complex proteomes, in- 23-min DIA method on average identified 33,909 peptides and cluding the human proteome. 3,413 distinct proteins in single measurements of six replicates One mechanism of maintaining proteome integrity is the (Q-value less than 1% at protein and precursor levels; Fig. 1 B marking and degradation of proteins that are damaged or no and C). This implies that ∼73% of proteins in the deep yeast longer needed with ubiquitin. The conjugation of ubiquitin to its spectral library were matched into the single runs. Note that the targets is catalyzed by E3 ubiquitin ligases, a diverse group of single runs represent only yeast grown in rich media (yeast extract enzymes that recognize and bind target proteins and facilitate peptone dextrose [YPD]), whereas the library combines the pro- ubiquitin transfer together with an E2, ubiquitin-conjugating teomes of yeast grown under several growth conditions and enzyme. Ubiquitination relies on a variety of cellular signals to therefore contains proteins which are not expressed during growth direct E3 ligases to their target proteins, and tight regulation of in YPD. Therefore, the degree of proteome completeness is likely this process is crucial for cellular viability (37). For instance, much higher than 73%. Measurements were highly reproducible during carbon starvation, yeast cells induce expression of the with Pearson coefficients greater than 0.92 between replicates (SI inactive GID (glucose-induced degradation) E3 ligase, which is Appendix,Fig.S1A) and coefficients of variation <20% for 68% of subsequently activated upon glucose replenishment. Following all common proteins between the six replicates. In comparison, a its activation, the GID E3 ligase targets gluconeogenic enzymes, single-run, data-dependent acquisition strategy with the same LC leading to their degradation and sparing the yeast from ener- gradient quantified only 11,883 peptides and 2,289 distinct pro- getically costly metabolic pathways that are unnecessary in fer- teins on average (Fig. 1 B and C). To more directly compare the mentable carbon sources (38–40). In addition, ubiquitin ligases performance of the 23-min DIA method to the DDA method we also serve as crucial regulators in response to oxidative, heavy analyzed the same sample with increasing
Recommended publications
  • Genetic Analysis of Retinopathy in Type 1 Diabetes

    Genetic Analysis of Retinopathy in Type 1 Diabetes

    Genetic Analysis of Retinopathy in Type 1 Diabetes by Sayed Mohsen Hosseini A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Institute of Medical Science University of Toronto © Copyright by S. Mohsen Hosseini 2014 Genetic Analysis of Retinopathy in Type 1 Diabetes Sayed Mohsen Hosseini Doctor of Philosophy Institute of Medical Science University of Toronto 2014 Abstract Diabetic retinopathy (DR) is a leading cause of blindness worldwide. Several lines of evidence suggest a genetic contribution to the risk of DR; however, no genetic variant has shown convincing association with DR in genome-wide association studies (GWAS). To identify common polymorphisms associated with DR, meta-GWAS were performed in three type 1 diabetes cohorts of White subjects: Diabetes Complications and Control Trial (DCCT, n=1304), Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR, n=603) and Renin-Angiotensin System Study (RASS, n=239). Severe (SDR) and mild (MDR) retinopathy outcomes were defined based on repeated fundus photographs in each study graded for retinopathy severity on the Early Treatment Diabetic Retinopathy Study (ETDRS) scale. Multivariable models accounted for glycemia (measured by A1C), diabetes duration and other relevant covariates in the association analyses of additive genotypes with SDR and MDR. Fixed-effects meta- analysis was used to combine the results of GWAS performed separately in WESDR, ii RASS and subgroups of DCCT, defined by cohort and treatment group. Top association signals were prioritized for replication, based on previous supporting knowledge from the literature, followed by replication in three independent white T1D studies: Genesis-GeneDiab (n=502), Steno (n=936) and FinnDiane (n=2194).
  • NIH Public Access Author Manuscript Science

    NIH Public Access Author Manuscript Science

    NIH Public Access Author Manuscript Science. Author manuscript; available in PMC 2014 September 08. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Science. 2014 January 31; 343(6170): 506–511. doi:10.1126/science.1247363. Exome Sequencing Links Corticospinal Motor Neuron Disease to Common Neurodegenerative Disorders A full list of authors and affiliations appears at the end of the article. # These authors contributed equally to this work. Abstract Hereditary spastic paraplegias (HSPs) are neurodegenerative motor neuron diseases characterized by progressive age-dependent loss of corticospinal motor tract function. Although the genetic basis is partly understood, only a fraction of cases can receive a genetic diagnosis, and a global view of HSP is lacking. By using whole-exome sequencing in combination with network analysis, we identified 18 previously unknown putative HSP genes and validated nearly all of these genes functionally or genetically. The pathways highlighted by these mutations link HSP to cellular transport, nucleotide metabolism, and synapse and axon development. Network analysis revealed a host of further candidate genes, of which three were mutated in our cohort. Our analysis links HSP to other neurodegenerative disorders and can facilitate gene discovery and mechanistic understanding of disease. Hereditary spastic paraplegias (HSPs) are a group of genetically heterogeneous neurodegenerative disorders with prevalence between 3 and 10 per 100,000 individuals (1). Hallmark features are axonal degeneration and progressive lower limb spasticity resulting from a loss of corticospinal tract (CST) function. HSP is classified into two broad categories, uncomplicated and complicated, on the basis of the presence of additional clinical features such as intellectual disability, seizures, ataxia, peripheral neuropathy, skin abnormalities, and visual defects.
  • Towards Relating the Evolution of the Gene Repertoire in Mammals to Tissue Specialisation

    Towards Relating the Evolution of the Gene Repertoire in Mammals to Tissue Specialisation

    Towards Relating the Evolution of the Gene Repertoire in Mammals to Tissue Specialisation Shiri Freilich Wolfson College This dissertation is submitted to the University of Cambridge for the degree of Doctor of Philosophy 21 December 2006 To Leon, who was the wind blowing in my sails, in the deep blue sea of this journey of ours. This Thesis is the result of my own work and includes nothing which is the outcome of work done in collaboration except where specifically indicated in the text. This Thesis does not exceed the specified length limit of 300 pages as defined by the Biology Degree Committee. This Thesis has been typeset in 12pt font according to the specifications defined by the Board of Graduate Studies and the Biology Degree Committee. 3 Summary: Towards Relating the Evolution of the Gene Repertoire in Mammals to Tissue Specialisation The sequencing efforts of recent years have provided a rich source of data for investigating how gene content determines similarity and uniqueness in a species’ phenotype. Work described in this PhD Thesis attempts to relate innovations in the gene repertoire along the mammalian lineage to the most obvious phenotypic characteristic of animals: the appearance of highly differentiated tissue types. Several different approaches, outlined below, have been followed to address some aspects of this problem. Initially, a comprehensive study of the pattern of expansion of the complement of enzymes in various species was performed in order to obtain a better view of the principles underlying the expansion of the gene repertoire in mammals. Although several studies have described a tendency toward an increase in sequence redundancy in mammals, not much is known about the way such sequence redundancy reflects functional redundancy.
  • Constraints and Consequences of the Emergence of Amino Acid Repeats in Eukaryotic Proteins

    Constraints and Consequences of the Emergence of Amino Acid Repeats in Eukaryotic Proteins

    ARTICLES Constraints and consequences of the emergence of amino acid repeats in eukaryotic proteins Sreenivas Chavali1 , Pavithra L Chavali1,2,5, Guilhem Chalancon1,5, Natalia Sanchez de Groot1, Rita Gemayel3,4, Natasha S Latysheva1, Elizabeth Ing-Simmons1, Kevin J Verstrepen3,4, Santhanam Balaji1 & M Madan Babu1 Proteins with amino acid homorepeats have the potential to be detrimental to cells and are often associated with human diseases. Why, then, are homorepeats prevalent in eukaryotic proteomes? In yeast, homorepeats are enriched in proteins that are essential and pleiotropic and that buffer environmental insults. The presence of homorepeats increases the functional versatility of proteins by mediating protein interactions and facilitating spatial organization in a repeat-dependent manner. During evolution, homorepeats are preferentially retained in proteins with stringent proteostasis, which might minimize repeat-associated detrimental effects such as unregulated phase separation and protein aggregation. Their presence facilitates rapid protein divergence through accumulation of amino acid substitutions, which often affect linear motifs and post-translational-modification sites. These substitutions may result in rewiring protein interaction and signaling networks. Thus, homorepeats are distinct modules that are often retained in stringently regulated proteins. Their presence facilitates rapid exploration of the genotype– phenotype landscape of a population, thereby contributing to adaptation and fitness. Repetitive sequences are
  • The Transition from Primary Colorectal Cancer to Isolated Peritoneal Malignancy

    The Transition from Primary Colorectal Cancer to Isolated Peritoneal Malignancy

    medRxiv preprint doi: https://doi.org/10.1101/2020.02.24.20027318; this version posted February 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . The transition from primary colorectal cancer to isolated peritoneal malignancy is associated with a hypermutant, hypermethylated state Sally Hallam1, Joanne Stockton1, Claire Bryer1, Celina Whalley1, Valerie Pestinger1, Haney Youssef1, Andrew D Beggs1 1 = Surgical Research Laboratory, Institute of Cancer & Genomic Science, University of Birmingham, B15 2TT. Correspondence to: Andrew Beggs, [email protected] KEYWORDS: Colorectal cancer, peritoneal metastasis ABBREVIATIONS: Colorectal cancer (CRC), Colorectal peritoneal metastasis (CPM), Cytoreductive surgery and heated intraperitoneal chemotherapy (CRS & HIPEC), Disease free survival (DFS), Differentially methylated regions (DMR), Overall survival (OS), TableFormalin fixed paraffin embedded (FFPE), Hepatocellular carcinoma (HCC) ARTICLE CATEGORY: Research article NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. 1 medRxiv preprint doi: https://doi.org/10.1101/2020.02.24.20027318; this version posted February 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . NOVELTY AND IMPACT: Colorectal peritoneal metastasis (CPM) are associated with limited and variable survival despite patient selection using known prognostic factors and optimal currently available treatments.
  • Role and Regulation of the P53-Homolog P73 in the Transformation of Normal Human Fibroblasts

    Role and Regulation of the P53-Homolog P73 in the Transformation of Normal Human Fibroblasts

    Role and regulation of the p53-homolog p73 in the transformation of normal human fibroblasts Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Lars Hofmann aus Aschaffenburg Würzburg 2007 Eingereicht am Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. Dr. Martin J. Müller Gutachter: Prof. Dr. Michael P. Schön Gutachter : Prof. Dr. Georg Krohne Tag des Promotionskolloquiums: Doktorurkunde ausgehändigt am Erklärung Hiermit erkläre ich, dass ich die vorliegende Arbeit selbständig angefertigt und keine anderen als die angegebenen Hilfsmittel und Quellen verwendet habe. Diese Arbeit wurde weder in gleicher noch in ähnlicher Form in einem anderen Prüfungsverfahren vorgelegt. Ich habe früher, außer den mit dem Zulassungsgesuch urkundlichen Graden, keine weiteren akademischen Grade erworben und zu erwerben gesucht. Würzburg, Lars Hofmann Content SUMMARY ................................................................................................................ IV ZUSAMMENFASSUNG ............................................................................................. V 1. INTRODUCTION ................................................................................................. 1 1.1. Molecular basics of cancer .......................................................................................... 1 1.2. Early research on tumorigenesis ................................................................................. 3 1.3. Developing
  • Hereditary Spastic Paraplegia: from Genes, Cells and Networks to Novel Pathways for Drug Discovery

    Hereditary Spastic Paraplegia: from Genes, Cells and Networks to Novel Pathways for Drug Discovery

    brain sciences Review Hereditary Spastic Paraplegia: From Genes, Cells and Networks to Novel Pathways for Drug Discovery Alan Mackay-Sim Griffith Institute for Drug Discovery, Griffith University, Brisbane, QLD 4111, Australia; a.mackay-sim@griffith.edu.au Abstract: Hereditary spastic paraplegia (HSP) is a diverse group of Mendelian genetic disorders affect- ing the upper motor neurons, specifically degeneration of their distal axons in the corticospinal tract. Currently, there are 80 genes or genomic loci (genomic regions for which the causative gene has not been identified) associated with HSP diagnosis. HSP is therefore genetically very heterogeneous. Finding treatments for the HSPs is a daunting task: a rare disease made rarer by so many causative genes and many potential mutations in those genes in individual patients. Personalized medicine through genetic correction may be possible, but impractical as a generalized treatment strategy. The ideal treatments would be small molecules that are effective for people with different causative mutations. This requires identification of disease-associated cell dysfunctions shared across geno- types despite the large number of HSP genes that suggest a wide diversity of molecular and cellular mechanisms. This review highlights the shared dysfunctional phenotypes in patient-derived cells from patients with different causative mutations and uses bioinformatic analyses of the HSP genes to identify novel cell functions as potential targets for future drug treatments for multiple genotypes. Keywords: neurodegeneration; motor neuron disease; spastic paraplegia; endoplasmic reticulum; Citation: Mackay-Sim, A. Hereditary protein-protein interaction network Spastic Paraplegia: From Genes, Cells and Networks to Novel Pathways for Drug Discovery. Brain Sci. 2021, 11, 403.
  • Expression of a Novel Reticulon-Like Gene in Human Testis

    Expression of a Novel Reticulon-Like Gene in Human Testis

    Reproduction (2002) 123, 227–234 Research Expression of a novel reticulon-like gene in human testis Z. M. Zhou1,2, J. H. Sha2, J. M. Li2 , M. Lin2, H. Zhu2, Y. D. Zhou2, L. R. Wang2, H. Zhu2, Y. Q. Wang1 and K. Y. Zhou1 1Institute of Genetic Resources, Nanjing Normal University, Nanjing, Jiangsu Province 210029, People’s Republic of China; and 2Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu Province, 210029, People’s Republic of China Identification of genes that are specifically expressed in the had 968 amino acids. This protein is homologous to the adult testis or the fetal testis is important for the study of six known members of the Rtn family (KIAA0886, Rtn xL, genes related to the development of the testis. In this study, reticulon 4a, Nogo-A, Nogo-A short form, and brain my043) a human testis cDNA microarray was established. PCR but was different at the 5’ end. All homologues originate products of 9216 clones from a human testis cDNA library from one gene, and result from both different promotor were dotted on a nylon membrane; mRNA from adult and regions and different splicing. Rtn-T lacks the first exon and fetal testes were purified and probes were prepared by a contains a second exon that is lacking in the other reverse transcription reaction with testis mRNA as template. homologues. Rtn-T is shorter than KIAA0886, Rtn xL, The microarray was hybridized with probes of adult and reticulon 4a and Nogo-A, but longer than the Nogo-A short fetal testes, and 96.8 and 95.4% of clones were positive, form and brain my043.
  • Leveraging Tissue Specific Gene Expression Regulation to Identify Genes Associated with Complex Diseases

    Leveraging Tissue Specific Gene Expression Regulation to Identify Genes Associated with Complex Diseases

    bioRxiv preprint doi: https://doi.org/10.1101/529297; this version posted January 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Leveraging tissue specific gene expression regulation to identify genes associated with complex diseases Wei Liu1,#, Mo Li2,#, Wenfeng Zhang2, Geyu Zhou1, Xing Wu4, Jiawei Wang1, Hongyu Zhao1,2,3* 1 Program of Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA 06510 2 Department of Biostatistics, Yale School of Public Health, New Haven, CT, USA 06510 3 Department of Genetics, Yale School of Medicine, New Haven, CT, USA 06510 4 Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA 06510 # These authors contributed equally to this work * To Whom correspondence should be addressed: Dr. Hongyu Zhao Department of Biostatistics Yale School of Public Health 60 College Street, New Haven, CT, 06520, USA [email protected] Key words: GWAS; gene expression imputation; Alzheimer’s disease; gene-level association test 1 bioRxiv preprint doi: https://doi.org/10.1101/529297; this version posted January 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract To increase statistical power to identify genes associated with complex traits, a number of methods like PrediXcan and FUSION have been developed using gene expression as a mediating trait linking genetic variations and diseases.
  • Super-Assembly of ER-Phagy Receptor Atg40 Induces Local ER Remodeling at Contacts with Forming Autophagosomal Membranes

    Super-Assembly of ER-Phagy Receptor Atg40 Induces Local ER Remodeling at Contacts with Forming Autophagosomal Membranes

    ARTICLE https://doi.org/10.1038/s41467-020-17163-y OPEN Super-assembly of ER-phagy receptor Atg40 induces local ER remodeling at contacts with forming autophagosomal membranes ✉ Keisuke Mochida1,4, Akinori Yamasaki 2,3,4, Kazuaki Matoba 2, Hiromi Kirisako1, Nobuo N. Noda 2 & ✉ Hitoshi Nakatogawa 1 1234567890():,; The endoplasmic reticulum (ER) is selectively degraded by autophagy (ER-phagy) through proteins called ER-phagy receptors. In Saccharomyces cerevisiae, Atg40 acts as an ER-phagy receptor to sequester ER fragments into autophagosomes by binding Atg8 on forming autophagosomal membranes. During ER-phagy, parts of the ER are morphologically rear- ranged, fragmented, and loaded into autophagosomes, but the mechanism remains poorly understood. Here we find that Atg40 molecules assemble in the ER membrane concurrently with autophagosome formation via multivalent interaction with Atg8. Atg8-mediated super- assembly of Atg40 generates highly-curved ER regions, depending on its reticulon-like domain, and supports packing of these regions into autophagosomes. Moreover, tight binding of Atg40 to Atg8 is achieved by a short helix C-terminal to the Atg8-family interacting motif, and this feature is also observed for mammalian ER-phagy receptors. Thus, this study sig- nificantly advances our understanding of the mechanisms of ER-phagy and also provides insights into organelle fragmentation in selective autophagy of other organelles. 1 School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Japan. 2 Institute of Microbial
  • Download Ppis for Each Single Seed, Thus Obtaining Each Seed’S Interactome (Ferrari Et Al., 2018)

    Download Ppis for Each Single Seed, Thus Obtaining Each Seed’S Interactome (Ferrari Et Al., 2018)

    bioRxiv preprint doi: https://doi.org/10.1101/2021.01.14.425874; this version posted January 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Integrating protein networks and machine learning for disease stratification in the Hereditary Spastic Paraplegias Nikoleta Vavouraki1,2, James E. Tomkins1, Eleanna Kara3, Henry Houlden3, John Hardy4, Marcus J. Tindall2,5, Patrick A. Lewis1,4,6, Claudia Manzoni1,7* Author Affiliations 1: Department of Pharmacy, University of Reading, Reading, RG6 6AH, United Kingdom 2: Department of Mathematics and Statistics, University of Reading, Reading, RG6 6AH, United Kingdom 3: Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, London, WC1N 3BG, United Kingdom 4: Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, WC1N 3BG, United Kingdom 5: Institute of Cardiovascular and Metabolic Research, University of Reading, Reading, RG6 6AS, United Kingdom 6: Department of Comparative Biomedical Sciences, Royal Veterinary College, London, NW1 0TU, United Kingdom 7: School of Pharmacy, University College London, London, WC1N 1AX, United Kingdom *Corresponding author: [email protected] Abstract The Hereditary Spastic Paraplegias are a group of neurodegenerative diseases characterized by spasticity and weakness in the lower body. Despite the identification of causative mutations in over 70 genes, the molecular aetiology remains unclear. Due to the combination of genetic diversity and variable clinical presentation, the Hereditary Spastic Paraplegias are a strong candidate for protein- protein interaction network analysis as a tool to understand disease mechanism(s) and to aid functional stratification of phenotypes.
  • The Effects of ER Morphology on Synaptic Structure and Function in Drosophila Melanogaster James B

    The Effects of ER Morphology on Synaptic Structure and Function in Drosophila Melanogaster James B

    © 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 1635-1648 doi:10.1242/jcs.184929 RESEARCH ARTICLE The effects of ER morphology on synaptic structure and function in Drosophila melanogaster James B. Summerville1,*, Joseph F. Faust1,*, Ethan Fan1, Diana Pendin2, Andrea Daga3, Joseph Formella1, Michael Stern1,‡ and James A. McNew1,‡ ABSTRACT (Di Sano et al., 2012). The nature of most of these secondary Hereditary spastic paraplegia (HSP) is a set of genetic diseases functions remains to be revealed. caused by mutations in one of 72 genes that results in age-dependent The large ER network also maintains luminal and membrane corticospinal axon degeneration accompanied by spasticity and continuity throughout the cytoplasm. This interconnected nature paralysis. Two genes implicated in HSPs encode proteins that of the ER network is required for ER function and is maintained regulate endoplasmic reticulum (ER) morphology. Atlastin 1 (ATL1, by the ER membrane fusion GTPase atlastin (Orso et al., 2009), also known as SPG3A) encodes an ER membrane fusion GTPase which is a member of the fusion dynamin-related protein family and reticulon 2 (RTN2, also known as SPG12) helps shape ER tube (fusion DRP) (McNew et al., 2013; Moss et al., 2011; Pendin et al., formation. Here, we use a new fluorescent ER marker to show that the 2011). ER within wild-type Drosophila motor nerve terminals forms a network The ER is closely associated with and functionally connected to of tubules that is fragmented and made diffuse upon loss of the the plasma membrane. This connection is often associated with 2+ atlastin 1 ortholog atl.