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Protein Trafficking In The Endosomal-Lysosomal System

内体-溶酶体系统中的蛋白质运输

基本信息

批准号：
10898489
负责人：
JUAN BONIFACINO
金额：
$ 335.83万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10898489
关键词：
ADP-Ribosylation Factor 1 Adaptor Protein Complex Subunits Adaptor Signaling Protein Address Architecture Attenuated Automobile Driving Autophagocytosis Biochemical Biogenesis Biotechnology Blood Coagulation Disorders Boston Capsid Proteins Cell membrane Cells Central Nervous System Childhood Clinical Trials Collaborations Complex Congenital Abnormality Congenital Disorders Cytoplasm Cytoplasmic Granules Defect Development Developmental Delay Disorders Disease Dynein ATPase Endocytosis Endoplasmic Reticulum Endosomes Enzymes Epithelial Cells Eukaryotic Cell Fibroblasts Genes Genetic Goals Golgi Apparatus HOP Pathway Hereditary Spastic Paraplegia Hermanski-Pudlak Syndrome Humulus Impairment Infection Integral Membrane Protein Intellectual functioning disability Intracellular Transport Intrathecal Injections Ion Channel Kinesin Knockout Mice Knowledge Laboratories Lead Lipids Lysosomes Mediating Medical center Melanosomes Membrane Membrane Protein Traffic Membrane Proteins Microtubules Molecular Molecular Chaperones Monomeric GTP-Binding Proteins Morphology Motor Movement Mus Muscle hypotonia Mutation Neurodegenerative Disorders Neurodevelopmental Disorder Neurons Organelles Pathogenesis Pathway interactions Patients Pediatric Hospitals Phenotype Phosphatidylinositols Physiological Processes Pigmentation Disorders Play Process Property Protein Sortings Proteins Protomer Rattus Recycling Regulation Reporting Research Role Safety Signal Transduction Site Sorting Source Spastic Paraplegia Swelling Syndrome System Tubular formation Tyrosine Universities Variant Vesicle Work base cytotoxic exosome fundamental research gene therapy infancy insight intercellular communication late endosome nervous system development nervous system disorder nonhuman primate pediatric patients polarized cell pre-clinical protein transport receptor recycling recruit retrograde transport stereotypy trans-Golgi Network vector

项目摘要

Our laboratory investigates the molecular mechanisms underlying the sorting of transmembrane proteins (known as cargo) to various compartments within the endomembrane system of eukaryotic cells, including the endoplasmic reticulum (ER), the Golgi apparatus, the trans-Golgi network (TGN), endosomes, lysosomes, lysosome-related organelles (LROs) (e.g., melanosomes, cytotoxic granules), and different domains of the plasma membrane in polarized cells, such as epithelial cells and neurons. Transport of cargo between these compartments is mediated by vesicular or tubular carriers that bud from a donor compartment, translocate through the cytoplasm, and fuse with an acceptor compartment. Our work focuses on the molecular machineries that mediate these processes in the context of different intracellular transport pathways, including endocytosis, recycling from endosomes to the plasma membrane, retrograde transport from endosomes to the TGN, biogenesis of lysosomes and LROs, autophagy, and polarized sorting in epithelial cells and neurons. Our fundamental research serves as a basis for elucidating disease mechanisms, particularly those related to congenital disorders of protein traffic, like the pigmentation and bleeding disorder Hermansky-Pudlak syndrome (HPS), hereditary spastic paraplegias (HSPs), and other neurodevelopmental and early infantile neurodegenerative disorders. The adaptor protein chaperone AAGAB promotes the assembly of the AP-4 complex - The adaptor protein 4 (AP-4) is a four-subunit complex that facilitates the export of specific transmembrane cargos, including autophagy protein 9A (ATG9A), from the trans-Golgi network (TGN). In recent years, mutations in any of the four AP-4 subunits were shown to cause a complicated form of hereditary spastic paraplegia known as "AP-4-deficiency syndrome." While previous studies provided insights into the role of AP-4 in cargo sorting within the cell, the process of AP-4 complex assembly remained elusive. This past year, we found that the AAGAB protein chaperone plays a critical role in the assembly of the AP-4 complex. Accordingly, AAGAB KO results in reduced levels of AP-4 subunits and accumulation of ATG9A at the TGN. These findings indicate that AP-4 assembly is not spontaneous but chaperone-assisted, deepening our understanding of the role of AP-4 in central nervous system development. Development of intrathecal AAV9/AP4M1 gene therapy for hereditary spastic paraplegia 50 caused by mutations in the mu4 subunit of AP-4 - Our laboratory also contributed to the development of gene therapy for hereditary spastic paraplegia 50 (SPG50) in collaboration with Xin Chen, Steven Gray (UT Southwestern Medical Center), Darius Ebrahimi-Fakhari (Boston Children's Hospital), and other colleagues. SPG50 is a childhood-onset neurological disorder caused by mutations in the AP4M1 gene encoding the mu4 subunit of AP-4. We demonstrated that infection of patients' fibroblasts with an AAV2/AP4M1 vector rescued the assembly of the AP-4 complex and the export of ATG9A from the trans-Golgi network. Our collaborators went on to show that intrathecal injection of an AAV9/AP4M1 vector had an acceptable safety profile in mice, rats, and non-human primates, and resulted in partial correction of phenotypic defects in AP4M1-KO mice. These preclinical results supported an investigational gene transfer clinical trial to treat SPG50. Architecture of the ESCPE-1 membrane coat: unveiling a key process of endosomal sorting - Intracellular recycling of membrane proteins plays a crucial role in the recycling of receptors, ion channels, and transporters. A critical player in this recycling machinery is the endosomal sorting complex for promoting exit 1 (ESCPE-1), responsible for rescuing transmembrane proteins from the endolysosomal pathway and transporting them to the trans-Golgi network and the plasma membrane. We collaborated with the laboratories of Aitor Hierro (CIC bioGUNE) and Daniel Castao-Dez (Biozentrum, University of Basel) to conduct biochemical, structural, and functional analyses into the organization of ESCPE-1, revealing that this complex forms a membrane coat with a single-layer architecture. Furthermore, we found that synergistic interactions between ESCPE-1 protomers, phosphoinositides, and cargo molecules lead to the global arrangement of amphipathic helices, driving the formation of recycling tubules. Endolysosome fusion attenuates exosome secretion - In previous research, we discovered an eight-subunit complex called BORC, which plays a crucial role in recruiting the small GTPase ARL8, kinesin motor proteins, and the tethering factor HOPS to late endosomes and lysosomes. We recently found that the BORC-ARL8-HOPS axis is responsible for regulating exosome secretion. Exosomes are small vesicles that cells release to dispose of undegraded materials and facilitate intercellular communication. A major source of exosomes is intraluminal vesicles within multivesicular endosomes, which can undergo fusion with either the plasma membrane or lysosomes. The factors that determine these alternative fates, however, were previously unknown. Our findings showed that disrupting the BORC-ARL8-HOPS axis impairs the fusion of late endosomes with lysosomes, thus increasing exosome secretion. These findings suggest that targeting the BORC-ARL8-HOPS pathway may be a promising strategy to enhance exosome yields for biotechnology applications. Small GTPases coordinate HOPS-mediated tethering of late endosomes and lysosomes - The transport of endocytosed cargoes to lysosomes relies on HOPS-dependent tethering of late endosomes to lysosomes prior to fusion. Although several small GTPases interact with HOPS, their exact localization and involvement in the tethering process remain unclear. To address this issue, we collaborated with Albert Haas and Andreas Jeschke (University of Bonn) to determine the order and functional interdependence of HOPS and its interacting proteins in cargo transport to lysosomes. Our findings revealed that the small GTPases RAB2A and RAB7 are associated with late endosomes, while the small GTPase ARL8 and its upstream regulator BORC localize to lysosomes. HOPS facilitates late endosome-lysosome fusion by bridging late endosomal RAB2A with lysosomal BORC-anchored ARL8. Additionally, we observed that RAB7 is not present at HOPS-dependent tethering sites but promotes fusion by facilitating the movement of late endosomes via dynein. ARF1-related disorder: unveiling pathophysiological mechanisms - ADP-ribosylation factor 1 (ARF1) is a small GTPase that plays a critical role in regulating membrane traffic at the Golgi apparatus and endosomes by interacting with various coat proteins and lipid-modifying enzymes. In collaboration with the laboratory of Tyler Pierson (Cedars-Sinai Medical Center), this past year, we reported a pediatric patient with an ARF1-related disorder caused by a monoallelic de novo missense variant (R99H) in ARF1. The patient presented with developmental delay, hypotonia, intellectual disability, and motor stereotypies. Our analyses showed that expression of R99H ARF1 led to swelling of the Golgi apparatus and increased recruitment of ARF1-regulated coat proteins, as well as altered the morphology of recycling endosomes. Furthermore, protein interaction analyses revealed that R99H ARF1 interacted more strongly with the ARF1-effector GGA3 compared to wild-type ARF1. These properties suggested that the pathogenetic mechanism of the R99H ARF1 variant involves constitutive activation, leading to Golgi and endosomal alterations. This study contributes to our understanding of the genetic basis of neurodevelopmental disorders associated with ARF1 variants.

项目成果

期刊论文数量（82）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Sorting of the Alzheimer's disease amyloid precursor protein mediated by the AP-4 complex.

DOI：
10.1016/j.devcel.2010.01.015
发表时间：
2010-03-16
期刊：
DEVELOPMENTAL CELL
影响因子：
11.8
作者：
Burgos, Patricia V.;Mardones, Gonzalo A.;Rojas, Adriana L.;daSilva, Luis L. P.;Prabhu, Yogikala;Hurley, James H.;Bonifacino, Juan S.
通讯作者：
Bonifacino, Juan S.

Inhibition of endolysosome fusion increases exosome secretion.

DOI：
10.1083/jcb.202209084
发表时间：
2023-06-05
期刊：
JOURNAL OF CELL BIOLOGY
影响因子：
7.8
作者：
Shelke, Ganesh Vilas;Williamson, Chad D.;Jarnik, Michal;Bonifacino, Juan S.
通讯作者：
Bonifacino, Juan S.

Vesicular transport earns a Nobel.

DOI：
10.1016/j.tcb.2013.11.001
发表时间：
2014-01-01
期刊：
Trends in cell biology
影响因子：
19
作者：
Bonifacino, Juan S
通讯作者：
Bonifacino, Juan S

Loss of endocytosis-associated RabGEF1 causes aberrant morphogenesis and altered autophagy in photoreceptors leading to retinal degeneration.

DOI：
10.1371/journal.pgen.1009259
发表时间：
2020-12
期刊：
PLoS genetics
影响因子：
4.5
作者：
Hargrove-Grimes P;Mondal AK;Gumerson J;Nellissery J;Aponte AM;Gieser L;Qian H;Fariss RN;Bonifacino JS;Li T;Swaroop A
通讯作者：
Swaroop A

α-Synuclein fibrils subvert lysosome structure and function for the propagation of protein misfolding between cells through tunneling nanotubes.

DOI：
10.1371/journal.pbio.3001287
发表时间：
2021-07
期刊：
PLoS biology
影响因子：
9.8
作者：
Dilsizoglu Senol A;Samarani M;Syan S;Guardia CM;Nonaka T;Liv N;Latour-Lambert P;Hasegawa M;Klumperman J;Bonifacino JS;Zurzolo C
通讯作者：
Zurzolo C