Publications by authors named "Stephen J Gould"

39 Publications

Exosome-mediated mRNA delivery in vivo is safe and can be used to induce SARS-CoV-2 immunity.

J Biol Chem 2021 11 1;297(5):101266. Epub 2021 Oct 1.

Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. Electronic address:

Functional delivery of mRNA has high clinical potential. Previous studies established that mRNAs can be delivered to cells in vitro and in vivo via RNA-loaded lipid nanoparticles (LNPs). Here we describe an alternative approach using exosomes, the only biologically normal nanovesicle. In contrast to LNPs, which elicited pronounced cellular toxicity, exosomes had no adverse effects in vitro or in vivo at any dose tested. Moreover, mRNA-loaded exosomes were characterized by efficient mRNA encapsulation (∼90%), high mRNA content, consistent size, and a polydispersity index under 0.2. Using an mRNA encoding the red light-emitting luciferase Antares2, we observed that mRNA-loaded exosomes were superior to mRNA-loaded LNPs at delivering functional mRNA into human cells in vitro. Injection of Antares2 mRNA-loaded exosomes also led to strong light emission following injection into the vitreous fluid of the eye or into the tissue of skeletal muscle in mice. Furthermore, we show that repeated injection of Antares2 mRNA-loaded exosomes drove sustained luciferase expression across six injections spanning at least 10 weeks, without evidence of signal attenuation or adverse injection site responses. Consistent with these findings, we observed that exosomes loaded with mRNAs encoding immunogenic forms of the SARS-CoV-2 Spike and Nucleocapsid proteins induced long-lasting cellular and humoral responses to both. Taken together, these results demonstrate that exosomes can be used to deliver functional mRNA to and into cells in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jbc.2021.101266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8483990PMC
November 2021

Extracellular vesicles derived from cardiosphere-derived cells as a potential antishock therapeutic.

J Trauma Acute Care Surg 2021 08;91(2S Suppl 2):S81-S88

From the Coagulation and Blood Research (Blood) (T.C.C., X.W., J.D.K., J.G.-M., C.L.S., B.L., A.P.C., J.A.B.), United States Army Institute of Surgical Research, San Antonio, Texas; Capricor Therapeutics Institute (J.J.M., K.A.P., L.R.-B., N.A.A., L.S.M.), Beverly Hills, California; Department of Biological Chemistry (S.J.G.), Johns Hopkins, Baltimore, Maryland; and Department of Biomedical Engineering (C.R.R.), The University of Texas at San Antonio, San Antonio, Texas.

Background: Extracellular vesicles (EVs) isolated from cardiosphere-derived cells (CDC-EVs) are coming to light as a unique cell-free therapeutic. Because of their novelty, however, there still exist prominent gaps in knowledge regarding their therapeutic potential. Herein the therapeutic potential of CDC-EVs in a rat model of acute traumatic coagulopathy induced by multiple injuries and hemorrhagic shock is outlined.

Methods: Extracellular vesicle surface expression of procoagulant molecules (tissue factor and phosphatidylserine) was evaluated by flow cytometry. Extracellular vesicle thrombogenicity was tested using calibrated thrombogram, and clotting parameters were assessed using a flow-based adhesion model simulating blood flow over a collagen-expressing surface. The therapeutic efficacy of EVs was then determined in a rat model of acute traumatic coagulopathy induced by multiple injuries and hemorrhagic shock.

Results: Extracellular vesicles isolated from cardiosphere-derived cells are not functionally procoagulant and do not interfere with platelet function. In a rat model of multiple injuries and hemorrhagic shock, early administration of EVs significantly reduced the elevation of lactate and creatinine and did not significantly enhance coagulopathy in rats with acute traumatic coagulopathy.

Conclusion: The results of this study are of great relevance to the development of EV products for use in combat casualty care, as our studies show that CDC-EVs have the potential to be an antishock therapeutic if administered early. These results demonstrate that research using CDC-EVs in trauma care needs to be considered and expanded beyond their reported cardioprotective benefits.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/TA.0000000000003218DOI Listing
August 2021

Choice of selectable marker affects recombinant protein expression in cells and exosomes.

J Biol Chem 2021 07 27;297(1):100838. Epub 2021 May 27.

Department of Biological Chemistry, School of Medicine, Johns Hopkins University, Baltimore Maryland, USA. Electronic address:

Transgenic mammalian cells are used for numerous research, pharmaceutical, industrial, and clinical purposes, and dominant selectable markers are often used to enable the selection of transgenic cell lines. Using HEK293 cells, we show here that the choice of selectable marker gene has a significant impact on both the level of recombinant protein expression and the cell-to-cell variability in recombinant protein expression. Specifically, we observed that cell lines generated with the NeoR or BsdR selectable markers and selected in the antibiotics G418 or blasticidin, respectively, displayed the lowest level of recombinant protein expression as well as the greatest cell-to-cell variability in transgene expression. In contrast, cell lines generated with the BleoR marker and selected in zeocin yielded cell lines that expressed the highest levels of linked recombinant protein, approximately 10-fold higher than those selected using the NeoR or BsdR markers, as well as the lowest cell-to-cell variability in recombinant protein expression. Intermediate yet still-high levels of expression were observed in cells generated with the PuroR- or HygR-based vectors and that were selected in puromycin or hygromycin, respectively. Similar results were observed in the African green monkey cell line COS7. These data indicate that each combination of selectable marker and antibiotic establishes a threshold below which no cell can survive and that these thresholds vary significantly between different selectable markers. Moreover, we show that choice of selectable marker also affects recombinant protein expression in cell-derived exosomes, consistent with the hypothesis that exosome protein budding is a stochastic rather than determinative process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jbc.2021.100838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8258971PMC
July 2021

The D614G Mutation Enhances the Lysosomal Trafficking of SARS-CoV-2 Spike.

bioRxiv 2020 Dec 9. Epub 2020 Dec 9.

The spike D614G mutation increases SARS-CoV-2 infectivity, viral load, and transmission but the molecular mechanism underlying these effects remains unclear. We report here that spike is trafficked to lysosomes and that the D614G mutation enhances the lysosomal sorting of spike and the lysosomal accumulation of spike-positive punctae in SARS-CoV-2-infected cells. Spike trafficking to lysosomes is an endocytosis-independent, V-ATPase-dependent process, and spike-containing lysosomes drive lysosome clustering but display poor lysotracker labeling and reduced uptake of endocytosed materials. These results are consistent with a lysosomal pathway of coronavirus biogenesis and raise the possibility that a common mechanism may underly the D614G mutation's effects on spike protein trafficking in infected cells and the accelerated entry of SARS-CoV-2 into uninfected cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.12.08.417022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7743070PMC
December 2020

Exosomes.

Annu Rev Biochem 2019 06;88:487-514

Department of Biological Chemistry, Johns Hopkins University, Baltimore, Maryland 21205, USA; email:

Exosomes are small, single-membrane, secreted organelles of ∼30 to ∼200 nm in diameter that have the same topology as the cell and are enriched in selected proteins, lipids, nucleic acids, and glycoconjugates. Exosomes contain an array of membrane-associated, high-order oligomeric protein complexes, display pronounced molecular heterogeneity, and are created by budding at both plasma and endosome membranes. Exosome biogenesis is a mechanism of protein quality control, and once released, exosomes have activities as diverse as remodeling the extracellular matrix and transmitting signals and molecules to other cells. This pathway of intercellular vesicle traffic plays important roles in many aspects of human health and disease, including development, immunity, tissue homeostasis, cancer, and neurodegenerative diseases. In addition, viruses co-opt exosome biogenesis pathways both for assembling infectious particles and for establishing host permissiveness. On the basis of these and other properties, exosomes are being developed as therapeutic agents in multiple disease models.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1146/annurev-biochem-013118-111902DOI Listing
June 2019

Advances, challenges, and opportunities in extracellular RNA biology: insights from the NIH exRNA Strategic Workshop.

JCI Insight 2018 04 5;3(7). Epub 2018 Apr 5.

Cancer Immunology, Hematology, and Etiology Branch, Division of Cancer Biology, National Cancer Institute, Bethesda, Maryland, USA.

Extracellular RNA (exRNA) has emerged as an important transducer of intercellular communication. Advancing exRNA research promises to revolutionize biology and transform clinical practice. Recent efforts have led to cutting-edge research and expanded knowledge of this new paradigm in cell-to-cell crosstalk; however, gaps in our understanding of EV heterogeneity and exRNA diversity pose significant challenges for continued development of exRNA diagnostics and therapeutics. To unravel this complexity, the NIH convened expert teams to discuss the current state of the science, define the significant bottlenecks, and brainstorm potential solutions across the entire exRNA research field. The NIH Strategic Workshop on Extracellular RNA Transport helped identify mechanistic and clinical research opportunities for exRNA biology and provided recommendations on high priority areas of research that will advance the exRNA field.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.98942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5928855PMC
April 2018

Cell biology: Organelle formation from scratch.

Nature 2017 02 1;542(7640):174-175. Epub 2017 Feb 1.

Department of Biological Chemistry, Johns Hopkins University, Baltimore, Maryland 2120, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature21496DOI Listing
February 2017

Extracellular vesicles carry microRNA-195 to intrahepatic cholangiocarcinoma and improve survival in a rat model.

Hepatology 2017 02 29;65(2):501-514. Epub 2016 Aug 29.

Division of Gastroenterology and Hepatology, School of Medicine, The Johns Hopkins University, Baltimore, MD.

The cancer microenvironment plays a central role in cancer development, growth, and homeostasis. This paradigm suggests that cancer fibroblasts support cancers, probably in response to stimuli received from the cancer cells. We aimed at investigating whether extracellular vesicles (EVs) can shuttle microRNA (miR) species between cancer-associated fibroblasts (CAFs) and cancer cells. To this end, we extracted EVs according to published protocols. EVs were studied for their miR content by quantitative reverse-transcription polymerase chain reaction. EVs were transfected with select miR species and utilized in vitro as well as in vivo in a rat model of cholangiocarcinoma (CCA). We found that miR-195 is down-regulated in CCA cells, as well as in adjoining fibroblasts. Furthermore, we report that EVs shuttle miR-195 from fibroblasts to cancer cells. Last, we show that fibroblast-derived EVs, loaded with miR-195, can be administered in a rat model of CCA, concentrate within the tumor, decrease the size of cancers, and improve survival of treated rats.

Conclusion: EVs play a salient role in trafficking miR species between cancer cells and CAFs in human CCA. Understanding of these mechanisms may allow devising of novel therapeutics. (Hepatology 2017;65:501-514).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep.28735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5258762PMC
February 2017

As we wait: coping with an imperfect nomenclature for extracellular vesicles.

J Extracell Vesicles 2013 15;2. Epub 2013 Feb 15.

Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3402/jev.v2i0.20389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3760635PMC
September 2013

The cis-acting signals that target proteins to exosomes and microvesicles.

Biochem Soc Trans 2013 Feb;41(1):277-82

Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.

Proteins bud from cells in small single-membraned vesicles (~50-250 nm) that have the same topology as the cell. Known variously as exosomes and microvesicles (EMVs), these extracellular organelles are enriched for specific proteins, lipids, carbohydrates and nucleic acids. EMV biogenesis plays critical roles in protein quality control and cell polarity, and, once released, EMVs can transmit signals and molecules to neighbouring cells via a non-viral pathway of intercellular vesicle traffic. In the present paper, we discuss the cis-acting targeting signals that target proteins to EMVs and mediate protein budding from the cell.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BST20120275DOI Listing
February 2013

HIV Pol inhibits HIV budding and mediates the severe budding defect of Gag-Pol.

PLoS One 2012 3;7(1):e29421. Epub 2012 Jan 3.

Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

The prevailing hypothesis of HIV budding posits that the viral Gag protein drives budding, and that the Gag p6 peptide plays an essential role by recruiting host-cell budding factors to sites of HIV assembly. HIV also expresses a second Gag protein, p160 Gag-Pol, which lacks p6 and fails to bud from cells, consistent with the prevailing hypothesis of HIV budding. However, we show here that the severe budding defect of Gag-Pol is not caused by the absence of p6, but rather, by the presence of Pol. Specifically, we show that (i) the budding defect of Gag-Pol is unaffected by loss of HIV protease activity and is therefore an intrinsic property of the Gag-Pol polyprotein, (ii) the N-terminal 433 amino acids of Gag and Gag-Pol are sufficient to drive virus budding even though they lack p6, (iii) the severe budding defect of Gag-Pol is caused by a dominant, cis-acting inhibitor of budding in the HIV Pol domain, and (iv) Gag-Pol inhibits Gag and virus budding in trans, even at normal levels of Gag and Gag-Pol expression. These and other data support an alternative hypothesis of HIV budding as a process that is mediated by the normal, non-viral pathway of exosome/microvesicle biogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0029421PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3250436PMC
May 2012

Biogenesis of the posterior pole is mediated by the exosome/microvesicle protein-sorting pathway.

J Biol Chem 2011 Dec 24;286(51):44162-44176. Epub 2011 Aug 24.

Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205. Electronic address:

Biogenesis of the posterior pole is critical to directed cell migration and other polarity-dependent processes. We show here that proteins are targeted to the posterior pole on the basis of higher order oligomerization and plasma membrane binding, the same elements that target proteins to exosomes/microvesicles (EMVs), HIV, and other retrovirus particles. We also demonstrate that the polarization of the EMV protein-sorting pathway can occur in morphologically non-polarized cells, defines the site of uropod formation, is induced by increased expression of EMV cargo proteins, and is evolutionarily conserved between humans and the protozoan Dictyostelium discoideum. Based on these results, we propose a mechanism of posterior pole biogenesis in which elevated levels of EMV cargoes (i) polarize the EMV protein-sorting pathway, (ii) generate a nascent posterior pole, and (iii) prime cells for signal-induced biogenesis of a uropod. This model also offers a mechanistic explanation for the polarized budding of EMVs and retroviruses, including HIV.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M111.274803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3243529PMC
December 2011

Protein targeting to exosomes/microvesicles by plasma membrane anchors.

J Biol Chem 2011 Apr 7;286(16):14383-95. Epub 2011 Feb 7.

Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

Animal cells secrete small vesicles, otherwise known as exosomes and microvesicles (EMVs). A short, N-terminal acylation tag can target a highly oligomeric cytoplasmic protein, TyA, into secreted vesicles (Fang, Y., Wu, N., Gan, X., Yan, W., Morell, J. C., and Gould, S. J. (2007) PLoS Biol. 5, 1267-1283). However, it is not clear whether this is true for other membrane anchors or other highly oligomeric, cytoplasmic proteins. We show here that a variety of plasma membrane anchors can target TyA-GFP to sites of vesicle budding and into EMVs, including: (i) a myristoylation tag; (ii) a phosphatidylinositol-(4,5)-bisphosphate (PIP(2))-binding domain; (iii), a phosphatidylinositol-(3,4,5)-trisphosphate-binding domain; (iv) a prenylation/palmitoylation tag, and (v) a type-1 plasma membrane protein, CD43. However, the relative budding efficiency induced by these plasma membrane anchors varied over a 10-fold range, from 100% of control (AcylTyA-GFP) for the myristoylation tag and PIP(2)-binding domain, to one-third or less for the others, respectively. Targeting TyA-GFP to endosome membranes by fusion to a phosphatidylinositol 3-phosphate-binding domain induced only a slight budding of TyA-GFP, ∼2% of control, and no budding was observed when TyA-GFP was targeted to Golgi membranes via a phosphatidylinositol 4-phosphate-binding domain. We also found that a plasma membrane anchor can target two other highly oligomeric, cytoplasmic proteins to EMVs. These observations support the hypothesis that plasma membrane anchors can target highly oligomeric, cytoplasmic proteins to EMVs. Our data also provide additional parallels between EMV biogenesis and retrovirus budding, as the anchors that induced the greatest budding of TyA-GFP are the same as those that mediate retrovirus budding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M110.208660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3077638PMC
April 2011

Identification of an inhibitory budding signal that blocks the release of HIV particles and exosome/microvesicle proteins.

Mol Biol Cell 2011 Mar 19;22(6):817-30. Epub 2011 Jan 19.

Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Animal cells bud exosomes and microvesicles (EMVs) from endosome and plasma membranes. The combination of higher-order oligomerization and plasma membrane binding is a positive budding signal that targets diverse proteins into EMVs and retrovirus particles. Here we describe an inhibitory budding signal (IBS) from the human immunodeficiency virus (HIV) Gag protein. This IBS was identified in the spacer peptide 2 (SP2) domain of Gag, is activated by C-terminal exposure of SP2, and mediates the severe budding defect of p6-deficient and PTAP-deficient strains of HIV. This IBS also impairs the budding of CD63 and several other viral and nonviral EMV proteins. The IBS does not prevent cargo delivery to the plasma membrane, a major site of EMV and virus budding. However, the IBS does inhibit an interaction between EMV cargo proteins and VPS4B, a component of the endosomal sorting complexes required for transport (ESCRT) machinery. Taken together, these results demonstrate that inhibitory signals can block protein and virus budding, raise the possibility that the ESCRT machinery plays a role in EMV biogenesis, and shed new light on the role of the p6 domain and PTAP motif in the biogenesis of HIV particles.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E10-07-0625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3057706PMC
March 2011

Variation and uncertainties in the classification of sudden unexpected infant deaths among paediatric pathologists in the UK: findings of a National Delphi Study.

J Clin Pathol 2010 Sep;63(9):796-9

Department of Cellular Pathology, John Radcliffe Hospital, Oxford, UK.

Aims: Presently, pathologists in the UK use diverse terminologies for the classification of sudden unexpected infant deaths, including 'unascertained,' 'sudden unexpected death in infancy' (SUDI) and 'sudden infant death syndrome' (SIDS). This study uses the Delphi method to investigate the views of paediatric pathologists on their use of these terms in order to determine areas of consensus.

Methods: There were three Delphi rounds overall; in the final one, participants were asked to score each statement using a modified Likert scale (0-9). The scores were analysed using non-parametric statistics, and statements in which the median score was or=7 (approximately 70% agreement) were considered to have reached 'consensus agreement.'

Results: Twenty-five of the 36 UK paediatric pathologists who were approached in the initial round contributed to all three rounds. There was consensus that 'SIDS' be used for unexplained sudden unexpected infant deaths that occurred during sleep. 'Infancy' was defined as up to 1 year of age, but there was no consensus regarding the lower age limit of SIDS. There was agreement that 'SUDI' be used for unexplained sudden infant deaths with a history of preceding illness, deaths with minor histological abnormalities of uncertain significance and co-sleeping-associated deaths. Most paediatric pathologists used 'unascertained' for findings suspicious of a non-natural cause of death. There was consensus that co-sleeping-associated deaths should be classified as 'unascertained' if parents had consumed alcohol or used drugs in the preceding 24 h.

Conclusions: The areas of consensus relating to terminology around SUDI and SIDS should guide future use by pathologists. However, there remains a significant lack of agreement, suggesting that acceptable alternative terms be identified for infant deaths which remain unexplained following autopsy in whom there are no suspicious features; the authors propose that 'unexplained SUDI,' followed by a comment, may represent the most factually correct compromise.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/jcp.2010.079715DOI Listing
September 2010

Oleate beta-oxidation in yeast involves thioesterase but not Yor180c protein that is not a dienoyl-CoA isomerase.

Biochim Biophys Acta 2009 May;1791(5):371-8

Department of Chemistry, City College and Graduate School of the City University of New York, New York, NY 10031, USA.

The beta-oxidation of oleic acid in Saccharomyces cerevisiae (S. cerevisiae) was studied by comparing the growth of wild-type cells on oleic acid or palmitic acid with the growth of mutants that either had a deletion in the YOR180c (DCI1) gene reported to encode delta3,5,delta2,4-dienoyl-CoA isomerase (dienoyl-CoA isomerase) or in the PTE1 gene encoding peroxisomal thioesterase 1. Growth of wild-type cells was indistinguishable from that of YOR180c mutant cells on either palmitic acid or oleic acid, whereas the PTE1 mutant grew slower and to a lower density on oleic acid but not on palmitic acid. The identification of 3,5-tetradecadienoic acid in the medium of wild-type cells but not in the medium of the PTE1 mutant proves the operation of the thioesterase-dependent pathway of oleate beta-oxidation in S. cerevisiae. Dienoyl-CoA isomerase activity was very low in wild-type cells, fourfold higher in the YOR180c mutant, and not associated with purified Yor180c protein. These observations support the conclusion that the YOR180c gene does not encode dienoyl-CoA isomerase.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbalip.2009.01.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3005976PMC
May 2009

Higher-order oligomerization targets plasma membrane proteins and HIV gag to exosomes.

PLoS Biol 2007 Jun;5(6):e158

Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

Exosomes are secreted organelles that have the same topology as the cell and bud outward (outward is defined as away from the cytoplasm) from endosome membranes or endosome-like domains of plasma membrane. Here we describe an exosomal protein-sorting pathway in Jurkat T cells that selects cargo proteins on the basis of both higher-order oligomerization (the oligomerization of oligomers) and plasma membrane association, acts on proteins seemingly without regard to their function, sequence, topology, or mechanism of membrane association, and appears to operate independently of class E vacuolar protein-sorting (VPS) function. We also show that higher-order oligomerization is sufficient to target plasma membrane proteins to HIV virus-like particles, that diverse Gag proteins possess exosomal-sorting information, and that higher-order oligomerization is a primary determinant of HIV Gag budding/exosomal sorting. In addition, we provide evidence that both the HIV late domain and class E VPS function promote HIV budding by unexpectedly complex, seemingly indirect mechanisms. These results support the hypothesis that HIV and other retroviruses are generated by a normal, nonviral pathway of exosome biogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.0050158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1885833PMC
June 2007

Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane.

J Cell Biol 2006 Mar;172(6):923-35

Department of Biological Chemistry and 2Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Exosomes are secreted, single membrane organelles of approximately 100 nm diameter. Their biogenesis is typically thought to occur in a two-step process involving (1) outward vesicle budding at limiting membranes of endosomes (outward = away from the cytoplasm), which generates intralumenal vesicles, followed by (2) endosome-plasma membrane fusion, which releases these internal vesicles into the extracellular milieu as exosomes. In this study, we present evidence that certain cells, including Jurkat T cells, possess discrete domains of plasma membrane that are enriched for exosomal and endosomal proteins, retain the endosomal property of outward vesicle budding, and serve as sites of immediate exosome biogenesis. It has been hypothesized that retroviruses utilize the exosome biogenesis pathway for the formation of infectious particles. In support of this, we find that Jurkat T cells direct the key budding factor of HIV, HIV Gag, to these endosome-like domains of plasma membrane and secrete HIV Gag from the cell in exosomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.200508014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2063735PMC
March 2006

Assay and functional analysis of dynamin-like protein 1 in peroxisome division.

Methods Enzymol 2005 ;404:586-97

Department of Cell Biology and Cell Pathology, University of Marburg, Germany.

Recent studies have demonstrated that peroxisome division requires at least one dynamin-like protein, Vps1p, in the yeast Saccharomyces cerevisiae and DLP1 (DRP1) in mammalian cells. Although the requirement for these proteins in peroxisome division is supported by many lines of evidence, their roles in peroxisome division have yet to be identified. Given the independence of peroxisomes from other organelle systems, the peroxisome system appears to have unique attributes for studying the function of dynamin-like proteins in organelle division. Here, we present methods that have been used for studying the role of DLP1 in peroxisome biogenesis and division.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/S0076-6879(05)04051-6DOI Listing
March 2006

Alternative splicing suggests extended function of PEX26 in peroxisome biogenesis.

Am J Hum Genet 2005 Jun 27;76(6):987-1007. Epub 2005 Apr 27.

McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Matsumoto and colleagues recently identified PEX26 as the gene responsible for complementation group 8 of the peroxisome biogenesis disorders and showed that it encodes an integral peroxisomal membrane protein with a single C-terminal transmembrane domain and a cytosolic N-terminus that interacts with the PEX1/PEX6 heterodimer through direct binding to the latter. They proposed that PEX26 functions as the peroxisomal docking factor for the PEX1/PEX6 heterodimer. Here, we identify new PEX26 disease alleles, localize the PEX6-binding domain to the N-terminal half of the protein (aa 29-174), and show that, at the cellular level, PEX26 deficiency impairs peroxisomal import of both PTS1- and PTS2-targeted matrix proteins. Also, we find that PEX26 undergoes alternative splicing to produce several splice forms--including one, PEX26- delta ex5, that maintains frame and encodes an isoform lacking the transmembrane domain of full-length PEX26 (PEX26-FL). Despite its cytosolic location, PEX26- delta ex5 rescues peroxisome biogenesis in PEX26-deficient cells as efficiently as does PEX26-FL. To test our observation that a peroxisomal location is not required for PEX26 function, we made a chimeric protein (PEX26-Mito) with PEX26 as its N-terminus and the targeting segment of a mitochondrial outer membrane protein (OMP25) at its C-terminus. We found PEX26-Mito localized to the mitochondria and directed all detectable PEX6 and a fraction of PEX1 to this extraperoxisomal location; yet PEX26-Mito retains the full ability to rescue peroxisome biogenesis in PEX26-deficient cells. On the basis of these observations, we suggest that a peroxisomal localization of PEX26 and PEX6 is not required for their function and that the interaction of PEX6 with PEX1 is dynamic. This model predicts that, once activated in an extraperoxisomal location, PEX1 moves to the peroxisome and completes the function of the PEX1/6 heterodimer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1086/430637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1196456PMC
June 2005

The evolution of alloimmunity and the genesis of adaptive immunity.

Q Rev Biol 2004 Dec;79(4):359-82

Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

Infectious agents select for host immune responses that destroy infectious nonself yet maintain tolerance to self. Here we propose that retroviruses and other host-antigen associated pathogens (HAAPs) select for the genetic, biochemical, and cell biological properties of alloimmunity, also known as the histocompatibility or tissue rejection response. This hypothesis predicts the major observations regarding histocompatibility responses, including: (i) their existence in animals as diverse as sponges and humans; (ii) extreme polymorphism and balanced allele frequencies at histocompatibility loci, including the human MHC and blood group loci; (iii) the frequency dependent selection of histocompatibility alleles; (iv) the ancient age of many alloantigenic polymorphisms; (v) the high ratio of nonsynonymous mutations to synonymous mutations at histocompatibility loci; (vi) disassortative mating based on MHC alleles; (vii) the inability to explain the existence and continuing selection of histocompatibility alleles by other more conventional biochemical and genetic paradigms; and (viii) the susceptibility of HAAPs, particularly retroviruses such as HIV (human immunodeficiency virus), to histocompatibility reactions. In addition, the hypothesis that HAAPs select the forms and molecules of alloimmunity offers simple explanations for the evolution of histocompatibility systems over time, the initial selection of hypervariable immune mechanisms, and the genesis of adaptive immunity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1086/426088DOI Listing
December 2004

PEX3 functions as a PEX19 docking factor in the import of class I peroxisomal membrane proteins.

J Cell Biol 2004 Mar 8;164(6):863-75. Epub 2004 Mar 8.

Dept. of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205, USA.

PEX19 is a chaperone and import receptor for newly synthesized, class I peroxisomal membrane proteins (PMPs). PEX19 binds these PMPs in the cytoplasm and delivers them to the peroxisome for subsequent insertion into the peroxisome membrane, indicating that there may be a PEX19 docking factor in the peroxisome membrane. Here we show that PEX3 is required for PEX19 to dock at peroxisomes, interacts specifically with the docking domain of PEX19, and is required for recruitment of the PEX19 docking domain to peroxisomes. PEX3 is also sufficient to dock PEX19 at heterologous organelles and binds PEX19 via a conserved motif that is essential for this docking activity and for PEX3 function in general. Not surprisingly, transient inhibition of PEX3 abrogates class I PMP import but has no effect on class II PMP import or peroxisomal matrix protein import. Taken together, these results suggest that PEX3 plays a selective, essential, and direct role in PMP import as a docking factor for PEX19.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.200311131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2172291PMC
March 2004

PEX19 is a predominantly cytosolic chaperone and import receptor for class 1 peroxisomal membrane proteins.

J Cell Biol 2004 Jan;164(1):57-67

Dept. of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.

Integral peroxisomal membrane proteins (PMPs) are synthesized in the cytoplasm and imported posttranslationally. Here, we demonstrate that PEX19 binds and stabilizes newly synthesized PMPs in the cytosol, binds to multiple PMP targeting signals (mPTSs), interacts with the hydrophobic domains of PMP targeting signals, and is essential for PMP targeting and import. These results show that PEX19 functions as both a chaperone and an import receptor for newly synthesized PMPs. We also demonstrate the existence of two PMP import mechanisms and two classes of mPTSs: class 1 mPTSs, which are bound by PEX19 and imported in a PEX19-dependent manner, and class 2 mPTSs, which are not bound by PEX19 and mediate protein import independently of PEX19.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.200304111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2171958PMC
January 2004

Evidence that HIV budding in primary macrophages occurs through the exosome release pathway.

J Biol Chem 2003 Dec 14;278(52):52347-54. Epub 2003 Oct 14.

Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

Lipid rafts are specialized regions of cell membranes enriched in cholesterol and sphingolipids that are involved in immune activation and signaling. Studies in T-cells indicate that these membrane domains serve as sites for release of human immunodeficiency virus (HIV). By budding through lipid rafts in T-cells, HIV selectively incorporates raft markers and excludes non-raft proteins. This process has been well studied in T-cells, but it is unknown whether lipid rafts serve as budding sites for HIV in macrophages. Recently, we proposed a new model of retroviral biogenesis called the Trojan exosome hypothesis (Gould, S. J., Booth, A., and Hildreth, J. E. K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10592-10597). This model proposes that retroviruses coopt the existing cellular machinery for exosomal release. Here, we performed the first test designed to differentiate between the lipid raft hypothesis of retroviral biogenesis and the Trojan exosome hypothesis. Using macrophages, we examined the relative abundance of several host proteins on the cell surface, in lipid rafts, and on both HIV particles and exosomes derived from these cells. Our results show significant differences in the abundance of host proteins on the cell surface and in HIV. Moreover, our data demonstrate discordance in the abundance of some proteins in lipid rafts and in HIV. Finally, our data reveal a strong concordance between the host cell protein profile of exosomes and that of HIV. These results strongly support the Trojan exosome hypothesis and its prediction that retroviral budding represents exploitation of a pre-existing cellular pathway of intercellular vesicle trafficking.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M309009200DOI Listing
December 2003

Peroxisome biogenesis disorders.

Annu Rev Genomics Hum Genet 2003 ;4:165-211

McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

The peroxisome biogenesis disorders (PBDs) comprise 12 autosomal recessive complementation groups (CGs). The multisystem clinical phenotype varies widely in severity and results from disturbances in both development and metabolic homeostasis. Progress over the last several years has lead to identification of the genes responsible for all of these disorders and to a much improved understanding of the biogenesis and function of the peroxisome. Increasing availability of mouse models for these disorders offers hope for a better understanding of their pathophysiology and for development of therapies that might especially benefit patients at the milder end of the clinical phenotype.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1146/annurev.genom.4.070802.110424DOI Listing
December 2003

The Trojan exosome hypothesis.

Proc Natl Acad Sci U S A 2003 Sep 28;100(19):10592-7. Epub 2003 Aug 28.

Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

We propose that retroviruses exploit a cell-encoded pathway of intercellular vesicle traffic, exosome exchange, for both the biogenesis of retroviral particles and a low-efficiency but mechanistically important mode of infection. This Trojan exosome hypothesis reconciles current paradigms of retrovirus-directed transmission with the unique lipid composition of retroviral particles, the host cell proteins present in retroviral particles, the complex cell biology of retroviral release, and the ability of retroviruses to infect cells independently of Envelope protein-receptor interactions. An exosomal origin also predicts that retroviruses pose an unsolvable paradox for adaptive immune responses, that retroviral antigen vaccines are unlikely to provide prophylactic protection, and that alloimmunity is a central component of antiretroviral immunity. Finally, the Trojan exosome hypothesis has important implications for the fight against HIV and AIDS, including how to develop new antiretroviral therapies, assess the risk of retroviral infection, and generate effective antiretroviral vaccines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1831413100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC196848PMC
September 2003

The dynamin-like GTPase DLP1 is essential for peroxisome division and is recruited to peroxisomes in part by PEX11.

J Biol Chem 2003 May 4;278(19):17012-20. Epub 2003 Mar 4.

Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

Peroxisome division involves the conserved PEX11 peroxisomal membrane proteins and in yeast has been shown to require Vps1p, a dynamin-like protein. We show here that DLP1, the human homolog of the yeast DNM1 and VPS1 genes, plays an important role in peroxisome division in human cells. Disruption of DLP1 function by either RNA interference or overexpressing dominant negative DLP1 mutants causes a dramatic reduction in peroxisome abundance, although overexpression of functional DLP1 has no effect on peroxisome abundance. Overexpression of PEX11 induces peroxisome division in a multistep process involving elongation of preexisting peroxisomes followed by their division. We find that DLP1 is dispensable for the first phase of this process but essential for the second. Furthermore, we show that DLP1 associates with peroxisomes and that PEX11 overexpression recruits DLP1 to peroxisome membranes. However, we were unable to detect physical interaction between PEX11 and DLP1, and the stoichiometry of PEX11 and peroxisome-associated DLP1 was far less than 1:1. Based on these and other aspects, we propose that DLP1 performs an essential but transient role in peroxisome division and that PEX11 promotes peroxisome division by recruiting DLP1 to peroxisome membranes through an indirect mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M212031200DOI Listing
May 2003

Correlating structure and affinity for PEX5:PTS1 complexes.

Biochemistry 2003 Feb;42(6):1660-6

Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

Many proteins that are destined to reside within the lumen of the peroxisome contain the peroxisomal targeting signal-1 (PTS1), a C-terminal tripeptide approximating the consensus sequence -Ser-Lys-Leu-COO(-). The PTS1 is recognized by the tetratricopeptide repeat (TPR) domains of PEX5, a cytosolic receptor that cycles between the cytoplasm and the peroxisome. To gain insight into the energetics of PTS1 binding specificity and to correlate these with features from the recently determined structure of a PEX5:PTS1 complex, we used a fluorescence-based binding assay that enables the quantitation of the dissociation constants for PTS1-containing peptide complexes with the TPR region of human PEX5. Through application of this assay to a collection of pentapeptides containing different C-terminal tripeptide sequences, including both natural and unnatural amino acids, the thermodynamic effects of sequence variation were examined. PTS1 variants that correspond to known functional targeting signals bind to the PEX5 fragment with a change in the standard binding free energy within 1.8 kcal mol(-1) of that corresponding to the peptide ending with -Ser-Lys-Leu-COO(-). The results suggest that a binding energy threshold may determine the functionality of PTS1 sequences.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi027034zDOI Listing
February 2003

PEX5 binds the PTS1 independently of Hsp70 and the peroxin PEX12.

J Biol Chem 2003 Mar 26;278(10):7897-901. Epub 2002 Nov 26.

Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

Most peroxisomal enzymes are targeted to peroxisomes by virtue of a type-1 peroxisomal targeting signal (PTS1) at their extreme C terminus. PEX5 binds the PTS1 through its C-terminal 40-kDa tetratricopeptide repeat domain and is essential for import of PTS1-contining proteins into peroxisomes. Here we examined the PTS1-binding activity of purified, recombinant, full-length PEX5 using a fluorescence anisotropy-based assay. Like its C-terminal fragment, full-length tetrameric PEX5 exhibits high intrinsic affinity for the PTS1, with a K(d) of 35 nm for the peptide lissamine-Tyr-Gln-Ser-Lys-Leu-COO(-). The specificity of this interaction was demonstrated by the fact that PEX5 had no detectable affinity for a peptide in which the Lys was replaced with Glu, a substitution that inactivates PTS1 signals in vivo. Hsp70 has been found to regulate the affinity of PEX5 for a PTS1-containing protein, but we found that the kinetics of PEX5-PTS1 binding was unaffected by Hsp70, Hsp70 plus ATP, or Hsp70 plus ADP. In addition, we found that another protein known to interact with the PTS1-binding domain of PEX5, the PEX12 zinc RING domain, also had no discernable effect on PEX5-PTS1 binding kinetics. Taken together, these results suggest that the initial step in peroxisomal protein import, the recognition of enzymes by PEX5, is a relatively simple process and that Hsp70 most probably stimulates this process by catalyzing the folding of newly synthesized peroxisomal enzymes and/or enhancing the accessibility of their PTS1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M206651200DOI Listing
March 2003
-->