175 results on '"McPherson PS"'
Search Results
2. Modeling Parkinson's disease pathology in human dopaminergic neurons by sequential exposure to α-synuclein fibrils and proinflammatory cytokines.
- Author
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Bayati A, Ayoubi R, Aguila A, Zorca CE, Deyab G, Han C, Recinto SJ, Nguyen-Renou E, Rocha C, Maussion G, Luo W, Shlaifer I, Banks E, McDowell I, Del Cid Pellitero E, Ding XE, Sharif B, Séguéla P, Yaqubi M, Chen CX, You Z, Abdian N, McBride HM, Fon EA, Stratton JA, Durcan TM, Nahirney PC, and McPherson PS
- Abstract
Lewy bodies (LBs), α-synuclein-enriched intracellular inclusions, are a hallmark of Parkinson's disease (PD) pathology, yet a cellular model for LB formation remains elusive. Recent evidence indicates that immune dysfunction may contribute to the development of PD. In this study, we found that induced pluripotent stem cell (iPSC)-derived human dopaminergic (DA) neurons form LB-like inclusions after treatment with α-synuclein preformed fibrils (PFFs) but only when coupled to a model of immune challenge (interferon-γ or interleukin-1β treatment) or when co-cultured with activated microglia-like cells. Exposure to interferon-γ impairs lysosome function in DA neurons, contributing to LB formation. The knockdown of LAMP2 or the knockout of GBA in conjunction with PFF administration is sufficient for inclusion formation. Finally, we observed that the LB-like inclusions in iPSC-derived DA neurons are membrane bound, suggesting that they are not limited to the cytoplasmic compartment but may be formed due to dysfunctions in autophagy. Together, these data indicate that immune-triggered lysosomal dysfunction may contribute to the development of PD pathology., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)
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- 2024
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3. Alpha-synuclein, autophagy-lysosomal pathway, and Lewy bodies: Mutations, propagation, aggregation, and the formation of inclusions.
- Author
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Bayati A and McPherson PS
- Abstract
Research into the pathophysiology of Parkinson's disease (PD) is a fast-paced pursuit, with new findings about PD and other synucleinopathies being made each year. The involvement of various lysosomal proteins, such as TFEB, TMEM175, GBA, and LAMP1/2, marks the rising awareness about the importance of lysosomes in PD and other neurodegenerative disorders. This, along with recent developments regarding the involvement of microglia and the immune system in neurodegenerative diseases, has brought about a new era in neurodegeneration: the role of proinflammatory cytokines on the nervous system, and their downstream effects on mitochondria, lysosomal degradation, and autophagy. More effort is needed to understand the interplay between neuroimmunology and disease mechanisms, as many of the mechanisms remain enigmatic. α-synuclein, a key protein in PD and the main component of Lewy bodies, sits at the nexus between lysosomal degradation, autophagy, cellular stress, neuroimmunology, PD pathophysiology, and disease progression. This review revisits some fundamental knowledge about PD while capturing some of the latest trends in PD research, specifically as it relates to α-synuclein., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2024
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4. Loss of symmetric cell division of apical neural progenitors drives DENND5A-related developmental and epileptic encephalopathy.
- Author
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Banks E, Francis V, Lin SJ, Kharfallah F, Fonov V, Lévesque M, Han C, Kulasekaran G, Tuznik M, Bayati A, Al-Khater R, Alkuraya FS, Argyriou L, Babaei M, Bahlo M, Bakhshoodeh B, Barr E, Bartik L, Bassiony M, Bertrand M, Braun D, Buchert R, Budetta M, Cadieux-Dion M, Calame DG, Cope H, Cushing D, Efthymiou S, Elmaksoud MA, El Said HG, Froukh T, Gill HK, Gleeson JG, Gogoll L, Goh ES, Gowda VK, Haack TB, Hashem MO, Hauser S, Hoffman TL, Hogue JS, Hosokawa A, Houlden H, Huang K, Huynh S, Karimiani EG, Kaulfuß S, Korenke GC, Kritzer A, Lee H, Lupski JR, Marco EJ, McWalter K, Minassian A, Minassian BA, Murphy D, Neira-Fresneda J, Northrup H, Nyaga DM, Oehl-Jaschkowitz B, Osmond M, Person R, Pehlivan D, Petree C, Sadleir LG, Saunders C, Schoels L, Shashi V, Spillmann RC, Srinivasan VM, Torbati PN, Tos T, Zaki MS, Zhou D, Zweier C, Trempe JF, Durcan TM, Gan-Or Z, Avoli M, Alves C, Varshney GK, Maroofian R, Rudko DA, and McPherson PS
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- Animals, Female, Humans, Male, Mice, Cell Polarity, Disease Models, Animal, Guanine Nucleotide Exchange Factors metabolism, Guanine Nucleotide Exchange Factors genetics, Membrane Proteins metabolism, Membrane Proteins genetics, Neurogenesis genetics, Cell Division, Induced Pluripotent Stem Cells metabolism, Induced Pluripotent Stem Cells cytology, Neural Stem Cells metabolism, Neural Stem Cells cytology
- Abstract
Developmental and epileptic encephalopathies (DEEs) feature altered brain development, developmental delay and seizures, with seizures exacerbating developmental delay. Here we identify a cohort with biallelic variants in DENND5A, encoding a membrane trafficking protein, and develop animal models with phenotypes like the human syndrome. We demonstrate that DENND5A interacts with Pals1/MUPP1, components of the Crumbs apical polarity complex required for symmetrical division of neural progenitor cells. Human induced pluripotent stem cells lacking DENND5A fail to undergo symmetric cell division with an inherent propensity to differentiate into neurons. These phenotypes result from misalignment of the mitotic spindle in apical neural progenitors. Cells lacking DENND5A orient away from the proliferative apical domain surrounding the ventricles, biasing daughter cells towards a more fate-committed state, ultimately shortening the period of neurogenesis. This study provides a mechanism for DENND5A-related DEE that may be generalizable to other developmental conditions and provides variant-specific clinical information for physicians and families., (© 2024. The Author(s).)
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- 2024
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5. The identification of high-performing antibodies for Sequestosome-1 for use in Western blot, immunoprecipitation and immunofluorescence.
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Ayoubi R, Alshafie W, Shlaifer I, Southern K, McPherson PS, and Laflamme C
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- Humans, Antibodies immunology, Sequestosome-1 Protein immunology, Sequestosome-1 Protein metabolism, Fluorescent Antibody Technique methods, Immunoprecipitation methods, Blotting, Western
- Abstract
Sequestosome-1, encoded by the gene SQSTM1 , functions as a bridge between ubiquitinated proteins and the proteasome or autophagosome, thereby regulating protein degradation pathways. Loss of Sequestosome-1 is hypothesized to enhance neurodegeneration progression in several diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal disorders (FTD). Sequestosome-1 reproducible research would be facilitated with the availability of well-characterized anti-Sequestosome-1 antibodies. In this study, we characterized seventeen Sequestosome-1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs., Competing Interests: Competing interests: For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - Abcam- ABclonal -Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems –Thermo Fisher Scientific., (Copyright: © 2024 Ayoubi R et al.)
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- 2024
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6. A guide to selecting high-performing antibodies for RNA-binding protein TIA1 for use in Western Blot, immunoprecipitation and immunofluorescence.
- Author
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Fotouhi M, Worrall D, Ayoubi R, Southern K, McPherson PS, and Laflamme C
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- T-Cell Intracellular Antigen-1 genetics, Blotting, Western, Fluorescent Antibody Technique, Immunoprecipitation, RNA-Binding Proteins
- Abstract
A member of the RNA-binding protein family, T-cell intracellular antigen-1 (TIA1) regulates mRNA translation and splicing as well as cellular stress by promoting stress granule formation. Variants of the TIA1 gene have implications in neurogenerative disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Reproducible research on TIA1 would be enhanced with the availability of high-quality anti-TIA1 antibodies. In this study, we characterized twelve TIA1 commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs., Competing Interests: Competing interests: For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - Abcam- Aviva Systems Biology- Bio Techne- Cell Signalling Technology- Developmental Studies Hybridoma Bank- GeneTex- Horizon Discovery - Proteintech - Synaptic Systems -Thermo Fisher Scientific., (Copyright: © 2024 Fotouhi M et al.)
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- 2024
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7. A neurodevelopmental disorder associated with a loss-of-function missense mutation in RAB35.
- Author
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Aguila A, Salah S, Kulasekaran G, Shweiki M, Shaul-Lotan N, Mor-Shaked H, Daana M, Harel T, and McPherson PS
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- Female, Humans, Male, ADP-Ribosylation Factor 6, ADP-Ribosylation Factors genetics, ADP-Ribosylation Factors metabolism, Cell Line, Cilia metabolism, Cilia genetics, Cilia pathology, Cytokinesis genetics, Guanine Nucleotide Exchange Factors genetics, Guanine Nucleotide Exchange Factors metabolism, Loss of Function Mutation, Pedigree, Models, Molecular, Protein Structure, Tertiary, Mutation, Missense, Neurodevelopmental Disorders genetics, Neurodevelopmental Disorders metabolism, Neurodevelopmental Disorders pathology, rab GTP-Binding Proteins genetics, rab GTP-Binding Proteins metabolism
- Abstract
Rab35 (Ras-associated binding protein) is a small GTPase that regulates endosomal membrane trafficking and functions in cell polarity, cytokinesis, and growth factor signaling. Altered Rab35 function contributes to progression of glioblastoma, defects in primary cilia formation, and altered cytokinesis. Here, we report a pediatric patient with global developmental delay, hydrocephalus, a Dandy-Walker malformation, axial hypotonia with peripheral hypertonia, visual problems, and conductive hearing impairment. Exome sequencing identified a homozygous missense variant in the GTPase fold of RAB35 (c.80G>A; p.R27H) as the most likely candidate. Functional analysis of the R27H-Rab35 variant protein revealed enhanced interaction with its guanine-nucleotide exchange factor, DENND1A and decreased interaction with a known effector, MICAL1, indicating that the protein is in an inactive conformation. Cellular expression of the variant drives the activation of Arf6, a small GTPase under negative regulatory control of Rab35. Importantly, variant expression leads to delayed cytokinesis and altered length, number, and Arl13b composition of primary cilia, known factors in neurodevelopmental disease. Our findings provide evidence of altered Rab35 function as a causative factor of a neurodevelopmental disorder., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2024
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8. DENND6A links Arl8b to a Rab34/RILP/dynein complex, regulating lysosomal positioning and autophagy.
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Kumar R, Khan M, Francis V, Aguila A, Kulasekaran G, Banks E, and McPherson PS
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- Protein Binding, Lysosomes metabolism, Autophagy, Dyneins metabolism, Adaptor Proteins, Signal Transducing genetics, Adaptor Proteins, Signal Transducing metabolism, rab GTP-Binding Proteins metabolism
- Abstract
Lysosomes help maintain cellular proteostasis, and defects in lysosomal positioning and function can cause disease, including neurodegenerative disorders. The spatiotemporal distribution of lysosomes is regulated by small GTPases including Rabs, which are activated by guanine nucleotide exchange factors (GEFs). DENN domain proteins are the largest family of Rab GEFs. Using a cell-based assay, we screened DENND6A, a member of the DENN domain protein family against all known Rabs and identified it as a potential GEF for 20 Rabs, including Rab34. Here, we demonstrate that DENND6A activates Rab34, which recruits a RILP/dynein complex to lysosomes, promoting lysosome retrograde transport. Further, we identify DENND6A as an effector of Arl8b, a major regulatory GTPase on lysosomes. We demonstrate that Arl8b recruits DENND6A to peripheral lysosomes to activate Rab34 and initiate retrograde transport, regulating nutrient-dependent lysosomal juxtanuclear repositioning. Loss of DENND6A impairs autophagic flux. Our findings support a model whereby Arl8b/DENND6A/Rab34-dependent lysosomal retrograde trafficking controls autophagy., (© 2024. The Author(s).)
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- 2024
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9. A guide to selecting high-performing antibodies for Rab1A and Rab1B for use in Western Blot, immunoprecipitation and immunofluorescence.
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Ruíz Moleón V, Fotouhi M, Ayoubi R, González Bolívar S, Southern K, McPherson PS, and Laflamme C
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- Humans, Reproducibility of Results, Fluorescent Antibody Technique, Blotting, Western, Immunoprecipitation, Proteins, rab1 GTP-Binding Proteins
- Abstract
Rab1 is a highly conserved small GTPase that exists in humans as two isoforms: Rab1A and Rab1B, sharing 92% sequence identity. These proteins regulate vesicle trafficking between the endoplasmic reticulum (ER) and Golgi and within the Golgi stacks. Rab1A and Rab1B may be oncogenes, as they are frequently dysregulated in various human cancers. Moreover, they contribute to the progression of Parkinson's disease. The availability of high-quality antibodies specific for Rab1A or Rab1B is essential to understand the distinct functions of these Rab1 proteins in both health and diseaseand to enhance the reproducibility of research involving these proteins. In this study, we characterized seven antibodies targeting Rab1A and five antibodies targeting Rab1B for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a much larger, collaborative initiative seeking to address the antibody reproducibility issue by characterizing commercially available antibodies for human proteins and publishing the results openly as a valuable resource for the scientific community. While uses of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs., Competing Interests: Competing interests: For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - Abcam-Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems –Thermo Fisher Scientific., (Copyright: © 2023 Ruíz Moleón V et al.)
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- 2023
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10. Identification of high-performing antibodies for Vacuolar protein sorting-associated protein 35 (hVPS35) for use in Western Blot, immunoprecipitation and immunofluorescence.
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Ayoubi R, Fotouhi M, Southern K, McPherson PS, and Laflamme C
- Subjects
- Blotting, Western, Fluorescent Antibody Technique, Immunoprecipitation, Protein Transport, Antibodies
- Abstract
Vacuolar protein sorting-associated protein 35 is a subunit of the retromer complex, a vital constituent of the endosomal protein sorting pathway. The D620N mutation in the VPS35 gene has been reported to be linked to type 17 Parkinson's Disease progression, the exact molecular mechanism remains to be solved. The scientific community would benefit from the accessibility of validated and high-quality anti-hVPS35 antibodies. In this study, we characterized thirteen hVPS35 commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs., Competing Interests: Competing interests: For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - Abcam- Aviva Systems Biology- Bio Techne- Cell Signalling Technology- Developmental Studies Hybridoma Bank- GeneTex- Horizon Discovery - Proteintech - Synaptic Systems -Thermo Fisher Scientific., (Copyright: © 2023 Ayoubi R et al.)
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- 2023
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11. Identification of high-performing antibodies for Superoxide dismutase [Cu-Zn] 1 (SOD1) for use in Western blot, immunoprecipitation, and immunofluorescence.
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Ayoubi R, Alshafie W, You Z, Southern K, McPherson PS, and Laflamme C
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- Superoxide Dismutase-1 genetics, Superoxide Dismutase-1 metabolism, Reproducibility of Results, Blotting, Western, Immunoprecipitation, Fluorescent Antibody Technique, Zinc, Superoxide Dismutase genetics, Superoxide Dismutase metabolism, Antibodies
- Abstract
Superoxide dismutase [Cu-Zn] 1 (SOD1), is an antioxidant enzyme encoded by the gene SOD1 , responsible for regulating oxidative stress levels by sequestering free radicals. Identified as the first gene with mutations in Amyotrophic lateral sclerosis (ALS), SOD1 is a determinant for studying diseases of aging and neurodegeneration. With guidance on well-characterized anti-SOD1 antibodies, the reproducibility of SOD1 research would be enhanced. In this study, we characterized eleven SOD1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs., Competing Interests: Competing interests: For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - Abcam- ABclonal -Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems –Thermo Fisher Scientific., (Copyright: © 2023 Ayoubi R et al.)
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- 2023
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12. Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications.
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Ayoubi R, Ryan J, Biddle MS, Alshafie W, Fotouhi M, Bolivar SG, Moleon VR, Eckmann P, Worrall D, McDowell I, Southern K, Reintsch W, Durcan TM, Brown CM, Bandrowski A, Virk HS, Edwards AM, McPherson PS, and Laflamme C
- Abstract
Antibodies are critical reagents to detect and characterize proteins. It is commonly understood that many commercial antibodies do not recognize their intended targets, but information on the scope of the problem remains largely anecdotal, and as such, feasibility of the goal of at least one potent and specific antibody targeting each protein in a proteome cannot be assessed. Focusing on antibodies for human proteins, we have scaled a standardized characterization approach using parental and knockout cell lines (Laflamme et al., 2019) to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins. Side-by-side comparisons of all antibodies against each target, obtained from multiple commercial partners, demonstrates that: i) more than 50% of all antibodies failed in one or more tests, ii ) yet, ~50-75% of the protein set was covered by at least one high-performing antibody, depending on application, suggesting that coverage of human proteins by commercial antibodies is significant; and iii ) recombinant antibodies performed better than monoclonal or polyclonal antibodies. The hundreds of underperforming antibodies identified in this study were found to have been used in a large number of published articles, which should raise alarm. Encouragingly, more than half of the underperforming commercial antibodies were reassessed by the manufacturers, and many had alterations to their recommended usage or were removed from the market. This first such study helps demonstrate the scale of the antibody specificity problem but also suggests an efficient strategy toward achieving coverage of the human proteome; mine the existing commercial antibody repertoire, and use the data to focus new renewable antibody generation efforts., Competing Interests: Competing interests The authors declare no competing interests.
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- 2023
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13. Heed a decade of calls for antibody validation.
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Kahn RA, Virk HS, and McPherson PS
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- Reproducibility of Results, Reference Standards, Antibodies immunology, Antibodies pharmacology, Indicators and Reagents standards, Antibody Specificity
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- 2023
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14. The identification of high-performing antibodies for Coiled-coil-helix-coiled-coil-helix domain containing protein 10 (CHCHD10) for use in Western Blot, immunoprecipitation and immunofluorescence.
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Ayoubi R, Alshafie W, Southern K, McPherson PS, and Laflamme C
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- Humans, HEK293 Cells, Fluorescent Antibody Technique methods, Immunoprecipitation methods, Mitochondrial Proteins immunology, Blotting, Western, Antibodies immunology
- Abstract
CHCHD10 is a mitochondrial protein, implicated in the regulation of mitochondrial morphology and cristae structure, as well as the maintenance of mitochondrial DNA integrity. Recently discovered to be associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in its mutant form, the scientific community would benefit from the availability of validated anti-CHCHD10 antibodies. In this study, we characterized four CHCHD10 commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. As this study highlights high-performing antibodies for CHCHD10, we encourage readers to use it as a guide to select the most appropriate antibody for their specific needs., Competing Interests: Competing interests: For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - Abcam- Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems –Thermo Fisher Scientific., (Copyright: © 2023 Ayoubi R et al.)
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- 2023
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15. The identification of high-performing antibodies for Charged multivesicular body protein 2b for use in Western Blot, immunoprecipitation and immunofluorescence.
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Alshafie W, Fotouhi M, Ayoubi R, Shlaifer I, Southern K, McPherson PS, and Laflamme C
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- Humans, Multivesicular Bodies, Reproducibility of Results, Blotting, Western, Fluorescent Antibody Technique, Immunoprecipitation, Antibodies, Amyotrophic Lateral Sclerosis, Frontotemporal Dementia
- Abstract
Charged multivesicular body protein 2B is a subunit of the endosomal sorting complex required for transport III (ESRCT-III), a complex implicated in the lysosomal degradation pathway and formation of multivesicular bodies. Mutations to the CHMP2B gene can result in abnormal protein aggregates in neurons and is therefore predicted to be associated in neurodegenerative diseases, including across the ALS-FTD spectrum. Through our standardized experimental protocol which compares read-outs in knockout cell lines and isogenic parental controls, this study aims to enhance the reproducibility of research on this target by characterizing eight commercial antibodies against charged multivesicular body protein 2b using Western Blot, immunoprecipitation, and immunofluorescence. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs., Competing Interests: Competing interests: For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - Abcam-Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems –Thermo Fisher Scientific., (Copyright: © 2023 Alshafie W et al.)
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- 2023
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16. DENND2B activates Rab35 at the intercellular bridge, regulating cytokinetic abscission and tetraploidy.
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Kumar R, Francis V, Ioannou MS, Aguila A, Khan M, Banks E, Kulasekaran G, and McPherson PS
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- Humans, Actin Cytoskeleton metabolism, Endosomal Sorting Complexes Required for Transport metabolism, Guanine Nucleotide Exchange Factors metabolism, HeLa Cells, Microfilament Proteins metabolism, Mixed Function Oxygenases metabolism, Tetraploidy, Actins metabolism, Cytokinesis physiology, rab GTP-Binding Proteins metabolism, DNA-Binding Proteins metabolism
- Abstract
Cytokinesis relies on membrane trafficking pathways regulated by Rabs and guanine nucleotide exchange factors (GEFs). During cytokinesis, the intercellular cytokinetic bridge (ICB) connecting daughter cells undergoes abscission, which requires actin depolymerization. Rab35 recruits MICAL1 to oxidize and depolymerize actin filaments. We show that DENND2B, a protein linked to cancer and congenital disorders, functions as a Rab35 GEF, recruiting and activating Rab35 at the ICB. DENND2B's N-terminal region also interacts with an active form of Rab35, suggesting that DENND2B is both a Rab35 GEF and effector. Knockdown of DENND2B delays abscission, leading to multinucleated cells and filamentous actin (F-actin) accumulation at the ICB, impairing recruitment of ESCRT-III at the abscission site. Additionally, F-actin accumulation triggers the formation of a chromatin bridge, activating the NoCut/abscission checkpoint, and DENND2B knockdown activates Aurora B kinase, a hallmark of checkpoint activation. Thus, our study identifies DENND2B as a crucial player in cytokinetic abscission., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)
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- 2023
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17. The identification of high-performing antibodies for RNA-binding protein FUS for use in Western Blot, immunoprecipitation, and immunofluorescence.
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Alshalfie W, Fotouhi M, Ayoubi R, You Z, Southern K, McPherson PS, and Laflamme C
- Subjects
- Reproducibility of Results, Blotting, Western, Fluorescent Antibody Technique, Immunoprecipitation, RNA-Binding Proteins, RNA-Binding Protein FUS genetics, Antibodies genetics
- Abstract
RNA-binding protein Fused-in Sarcoma (FUS) plays an essential role in various cellular processes. Mutations in the C-terminal domain region, where the nuclear localization signal (NLS) is located, causes the redistribution of FUS from the nucleus to the cytoplasm. In neurons, neurotoxic aggregates are formed as a result, contributing to neurogenerative diseases. Well-characterized anti-FUS antibodies would enable the reproducibility of FUS research, thereby benefiting the scientific community. In this study, we characterized ten FUS commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs., Competing Interests: Competing interests: For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - Abcam- ABclonal -Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems -Thermo Fisher Scientific., (Copyright: © 2023 Alshalfie W et al.)
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- 2023
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- View/download PDF
18. The identification of high-performing antibodies for TDP-43 for use in Western Blot, immunoprecipitation and immunofluorescence.
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Worrall D, Ayoubi R, Fotouhi M, Southern K, McPherson PS, and Laflamme C
- Subjects
- Cell Line, Blotting, Western, Fluorescent Antibody Technique, Immunoprecipitation, DNA-Binding Proteins genetics
- Abstract
TAR DNA-binding protein 43 (TDP-43) is a DNA/RNA binding protein playing a critical role in the regulation of transcription, splicing and RNA stability. Mutations in TARDBP leading to aggregation, are suspected to be a characteristic feature of various neurogenerative diseases. The lack of well-characterized anti- TDP-43 antibodies acts as a barrier to establish reproducible TDP-43 research. In this study, we characterized eighteen TDP-43 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many well-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs., Competing Interests: Competing interests: For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - Abcam- ABclonal -Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems -Thermo Fisher Scientific., (Copyright: © 2023 Worrall D et al.)
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- 2023
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19. The identification of high-preforming antibodies for Ubiquilin-2 for use in Western Blot, immunoprecipitation, and immunofluorescence.
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McDowell I, Ayoubi R, Fotouhi M, Southern K, McPherson PS, and Laflamme C
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- Blotting, Western, Fluorescent Antibody Technique, Transcription Factors metabolism, Immunoprecipitation, Adaptor Proteins, Signal Transducing, Cell Cycle Proteins metabolism
- Abstract
Ubiquilin-2, a member of the ubiquilin protein family, plays a role in the regulation of various protein degradation pathways, and is mutated in some neurodegenerative diseases. Well-characterized anti-Ubiquilin-2 antibodies would advance reproducible research for Ubiquilin-2 and in turn, benefit the scientific community. In this study, we characterized ten Ubiquilin-2 commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs., Competing Interests: Competing interests: For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - Abcam- ABclonal -Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems -ThermoFisher Scientific., (Copyright: © 2023 McDowell I et al.)
- Published
- 2023
- Full Text
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20. The identification of high-performing antibodies for Profilin-1 for use in Western blot, immunoprecipitation and immunofluorescence.
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Ayoubi R, McDowell I, Fotouhi M, Southern K, McPherson PS, and Laflamme C
- Subjects
- Humans, Fluorescent Antibody Technique, Mutation, Antibodies genetics, Blotting, Western, Immunoprecipitation, Neurodegenerative Diseases
- Abstract
Profilin-1, a member of the Profilin family, is a ubiquitously expressed protein that controls actin polymerization in a concentration-dependent manner. As mutations in the Profilin-1 gene have potential implications in neurodegenerative disease progression, well-characterized anti-Profilin-1 antibodies would be beneficial to the scientific community. In this study, we characterized sixteen Profilin-1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence applications, using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs., Competing Interests: Competing interests: For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: – Abcam – ABclonal – Aviva Systems Biology – Bio-Techne – Cell Signaling Technology – Developmental Studies Hybridoma Bank – GeneTex – Horizon Discovery – Proteintech – Synaptic Systems –Thermo Fisher Scientific., (Copyright: © 2023 Ayoubi R et al.)
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- 2023
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21. The identification of high-performing antibodies for transmembrane protein 106B (TMEM106B) for use in Western blot, immunoprecipitation, and immunofluorescence.
- Author
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Ayoubi R, Fotouhi M, Southern K, Bhajiawala R, Fanti R, Prinos P, McPherson PS, and Laflamme C
- Subjects
- Reproducibility of Results, Blotting, Western, Fluorescent Antibody Technique, Immunoprecipitation, Membrane Proteins metabolism, Nerve Tissue Proteins genetics
- Abstract
Transmembrane protein 106B (TMEM106B), a protein that is localized to the lysosome, is genetically linked to many neurodegenerative diseases and forms fibrils in diseased brains. The reproducibility of TMEM106B research would be enhanced if the community had access to well-characterized anti-TMEM106B antibodies. In this study, we characterized six commercially available TMEM106B antibodies for their performance in Western blot, immunoprecipitation, and immunofluorescence, using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs., Competing Interests: Competing interests: For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - Abcam- ABclonal -Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems -ThermoFisher Scientific. The Structural Genomics Consortium receives funds from Bayer AG, Boehringer Ingelheim, Bristol Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute, the EU and EFPIA, Janssen, Merck KGaA (also known as EMD in Canada and the United States), Pfizer and Takeda., (Copyright: © 2023 Ayoubi R et al.)
- Published
- 2023
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22. Visualization of α-synuclein trafficking via nanogold labeling and electron microscopy.
- Author
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Bayati A, Luo W, Del Cid-Pellitero E, Fon EA, Durcan TM, and McPherson PS
- Subjects
- Microscopy, Electron, Cells, Cultured, alpha-Synuclein, Neurons
- Abstract
There is conflicting evidence regarding the mechanisms of α-synuclein internalization, and its trafficking itinerary following cellular entry remains largely unknown. To examine these issues, we describe steps for coupling α-synuclein preformed fibrils (PFFs) to nanogold beads and their subsequent characterization by electron microscopy (EM). Then we describe the uptake of conjugated PFFs by U2OS cells plated on Permanox 8-well chamber slides. This process eliminates the reliance on antibody specificity and the need to employ complex immunoEM staining protocols. For complete details on the use and execution of this protocol, please refer to Bayati et al. (2022).
1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2023
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23. Correction: The ARSACS disease protein sacsin controls lysosomal positioning and reformation by regulating microtubule dynamics.
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Francis V, Alshafie W, Kumar R, Girard M, Brais B, and McPherson PS
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- 2022
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24. Identification and Utilization of a Chemical Probe to Interrogate the Roles of PIKfyve in the Lifecycle of β-Coronaviruses.
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Drewry DH, Potjewyd FM, Bayati A, Smith JL, Dickmander RJ, Howell S, Taft-Benz S, Min SM, Hossain MA, Heise M, McPherson PS, Moorman NJ, and Axtman AD
- Subjects
- Antiviral Agents pharmacology, Morpholines, Phosphates, Phosphatidylinositols, Phosphoinositide-3 Kinase Inhibitors, Coronavirus, Phosphatidylinositol 3-Kinases
- Abstract
From a designed library of indolyl pyrimidinamines, we identified a highly potent and cell-active chemical probe ( 17 ) that inhibits phosphatidylinositol-3-phosphate 5-kinase (PIKfyve). Comprehensive evaluation of inhibitor selectivity confirmed that this PIKfyve probe demonstrates excellent kinome-wide selectivity. A structurally related indolyl pyrimidinamine ( 30 ) was characterized as a negative control that lacks PIKfyve inhibitory activity and exhibits exquisite selectivity when profiled broadly. Chemical probe 17 disrupts multiple phases of the lifecycle of β-coronaviruses: viral replication and viral entry. The diverse antiviral roles of PIKfyve have not been previously probed comprehensively in a single study or using the same compound set. Our scaffold is a distinct chemotype that lacks the canonical morpholine hinge-binder of classical lipid kinase inhibitors and has a non-overlapping kinase off-target profile with known PIKfyve inhibitors. Our chemical probe set can be used by the community to further characterize the role of PIKfyve in virology.
- Published
- 2022
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25. The ARSACS disease protein sacsin controls lysosomal positioning and reformation by regulating microtubule dynamics.
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Francis V, Alshafie W, Kumar R, Girard M, Brais B, and McPherson PS
- Subjects
- Amino Acid Sequence, Mutation, Heat-Shock Proteins genetics, Heat-Shock Proteins metabolism, Lysosomes genetics, Lysosomes metabolism, Microtubules genetics, Microtubules metabolism, Muscle Spasticity genetics, Muscle Spasticity metabolism, Spinocerebellar Ataxias congenital, Spinocerebellar Ataxias genetics, Spinocerebellar Ataxias metabolism
- Abstract
Autosomal recessive spastic ataxia of Charlevoix-Saguenay is a fatal brain disorder featuring cerebellar neurodegeneration leading to spasticity and ataxia. This disease is caused by mutations in the SACS gene that encodes sacsin, a massive 4579-amino acid protein with multiple modular domains. However, molecular details of the function of sacsin are not clear. Here, using live cell imaging and biochemistry, we demonstrate that sacsin binds to microtubules and regulates microtubule dynamics. Loss of sacsin function in various cell types, including knockdown and KO primary neurons and patient fibroblasts, leads to alterations in lysosomal transport, positioning, function, and reformation following autophagy. Each of these phenotypic changes is consistent with altered microtubule dynamics. We further show the effects of sacsin are mediated at least in part through interactions with JIP3, an adapter for microtubule motors. These data reveal a new function for sacsin that explains its previously reported roles and phenotypes., Competing Interests: Conflict of interest P. S. M. is a distinguished James McGill Professor and Fellow of the Royal Society of Canada. The authors declare that they have no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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26. Identification of highly specific antibodies for Serine/threonine-protein kinase TBK1 for use in immunoblot, immunoprecipitation and immunofluorescence.
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Alshafie W, Fotouhi M, Shlaifer I, Ayoubi R, Edwards AM, Durcan TM, McPherson PS, and Laflamme C
- Subjects
- Mutation, Fluorescent Antibody Technique, Immunoprecipitation, Threonine, Serine, Protein Serine-Threonine Kinases, Antibodies
- Abstract
TBK1 is a serine-threonine protein kinase that has been linked to a number of diseases including amyotrophic lateral sclerosis and frontotemporal dementia. Reproducible research on TBK1 has been hampered by the lack of well characterized antibodies. In this study, we characterized 11 commercial antibodies for TBK1 for use in immunoblot, immunofluorescence and immunoprecipitation, using an isogeneic knock-out cell line as a control. We identify antibodies that appear specific for all three applications but invite the readers to interpret the present findings based on their own scientific expertise and use this report as a guide to select the most appropriate antibody for their specific needs., Competing Interests: Competing interests: The laboratory of Peter S McPherson was awarded a Genomic Applications Partnership Program grant from Genome Canada in 2021. For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell lines providers. The partners provide antibodies and knockout lines to the McPherson laboratory at no cost. Partners are: -Abcam -Abclonal -Aviva Systems Biology -Bio-Techne -Cell Signaling Technology -Developmental Studies Hybridoma Bank -Genetex -Horizon Discovery -Proteintech -Synaptic Systems -Thermo Fisher Scientific, (Copyright: © 2022 Alshafie W et al.)
- Published
- 2022
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27. Rapid macropinocytic transfer of α-synuclein to lysosomes.
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Bayati A, Banks E, Han C, Luo W, Reintsch WE, Zorca CE, Shlaifer I, Del Cid Pellitero E, Vanderperre B, McBride HM, Fon EA, Durcan TM, and McPherson PS
- Subjects
- Endosomes metabolism, Humans, Lysosomes metabolism, alpha-Synuclein metabolism, Parkinson Disease metabolism, Synucleinopathies
- Abstract
The nervous system spread of alpha-synuclein fibrils is thought to cause Parkinson's disease (PD) and other synucleinopathies; however, the mechanisms underlying internalization and cellular spread are enigmatic. Here, we use confocal and superresolution microscopy, subcellular fractionation, and electron microscopy (EM) of immunogold-labeled α-synuclein preformed fibrils (PFFs) to demonstrate that this form of the protein undergoes rapid internalization and is targeted directly to lysosomes in as little as 2 min. Uptake of PFFs is disrupted by macropinocytic inhibitors and circumvents classical endosomal pathways. Immunogold-labeled PFFs are seen at the highly curved inward edge of membrane ruffles, in newly formed macropinosomes, in multivesicular bodies and in lysosomes. While most fibrils remain in lysosomes, a portion is transferred to neighboring naive cells along with markers of exosomes. These data indicate that PFFs use a unique internalization mechanism as a component of cell-to-cell propagation., Competing Interests: Declaration of interests The authors declare no competing interests., (Crown Copyright © 2022. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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28. Host Kinase CSNK2 is a Target for Inhibition of Pathogenic SARS-like β-Coronaviruses.
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Yang X, Dickmander RJ, Bayati A, Taft-Benz SA, Smith JL, Wells CI, Madden EA, Brown JW, Lenarcic EM, Yount BL Jr, Chang E, Axtman AD, Baric RS, Heise MT, McPherson PS, Moorman NJ, and Willson TM
- Subjects
- Animals, Antiviral Agents pharmacology, Humans, Mice, Virus Internalization, Coronavirus genetics, Coronavirus Infections
- Abstract
Inhibition of the protein kinase CSNK2 with any of 30 specific and selective inhibitors representing different chemotypes, blocked replication of pathogenic human, bat, and murine β-coronaviruses. The potency of in-cell CSNK2A target engagement across the set of inhibitors correlated with antiviral activity and genetic knockdown confirmed the essential role of the CSNK2 holoenzyme in β-coronavirus replication. Spike protein endocytosis was blocked by CSNK2A inhibition, indicating that antiviral activity was due in part to a suppression of viral entry. CSNK2A inhibition may be a viable target for the development of anti-SARS-like β-coronavirus drugs.
- Published
- 2022
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29. A cell-based GEF assay reveals new substrates for DENN domains and a role for DENND2B in primary ciliogenesis.
- Author
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Kumar R, Francis V, Kulasekaran G, Khan M, Armstrong GAB, and McPherson PS
- Abstract
Primary cilia are sensory antennae crucial for cell and organism development, and defects in their biogenesis cause ciliopathies. Ciliogenesis involves membrane trafficking mediated by small guanosine triphosphatases (GTPases) including Rabs, molecular switches activated by guanine nucleotide exchange factors (GEFs). The largest family of Rab GEFs is the DENN domain-bearing proteins. Here, we screen all 60 Rabs against two major DENN domain families using a cellular GEF assay, uncovering 19 novel DENN/Rab pairs. The screen reveals Rab10 as a substrate for DENND2B, a protein previously implicated in cancer and severe mental retardation. Through activation of Rab10, DENND2B represses the formation of primary cilia. Through a second pathway, DENND2B functions as a GEF for RhoA to control the length of primary cilia. This work thus identifies an unexpected diversity in DENN domain-mediated activation of Rabs, a previously unidentified non-Rab substrate for a DENN domain, and a new regulatory protein in primary ciliogenesis.
- Published
- 2022
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30. Host kinase CSNK2 is a target for inhibition of pathogenic β-coronaviruses including SARS-CoV-2.
- Author
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Yang X, Dickmander RJ, Bayati A, Taft-Benz SA, Smith JL, Wells CI, Madden EA, Brown JW, Lenarcic EM, Yount BL Jr, Chang E, Axtman AD, Baric RS, Heise MT, McPherson PS, Moorman NJ, and Willson TM
- Abstract
Inhibition of the protein kinase CSNK2 with any of 30 specific and selective inhibitors representing different chemotypes, blocked replication of pathogenic human and murine β-coronaviruses. The potency of in-cell CSNK2A target engagement across the set of inhibitors correlated with antiviral activity and genetic knockdown confirmed the essential role of the CSNK2 holoenzyme in β-coronavirus replication. Spike protein uptake was blocked by CSNK2A inhibition, indicating that antiviral activity was due in part to a suppression of viral entry. CSNK2A inhibition may be a viable target for development of new broad spectrum anti-β-coronavirus drugs.
- Published
- 2022
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- View/download PDF
31. Opinion: Independent third-party entities as a model for validation of commercial antibodies.
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Laflamme C, Edwards AM, Bandrowski AE, and McPherson PS
- Subjects
- Humans, Reproducibility of Results, Antibodies, Biomedical Research, Indicators and Reagents standards
- Abstract
A vast array of commercial antibodies covers a large percentage of human gene products, but determining which among them is most appropriate for any given application is challenging. This leads to use of non-specific antibodies that contributes to issues with reproducibility. It is our opinion that the community of scientists who use commercial antibodies in their biomedical research would benefit from third-party antibody characterization entities that use standardized operating procedures to assess and compare antibody performance. Ideally, such entities would follow the principles of open science, such that all antibodies against any given protein target would be tested in parallel, and all data generated released to the public domain without bias. Furthermore, there should be no financial incentive for the entity beyond cost-recovery. Such non-profit organizations, combined with other scientific efforts, could catalyse new discoveries by providing scientists with better validated antibody tools., (Copyright © 2021 Elsevier B.V. All rights reserved.)
- Published
- 2021
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32. Early defects in mucopolysaccharidosis type IIIC disrupt excitatory synaptic transmission.
- Author
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Pará C, Bose P, Bruno L, Freemantle E, Taherzadeh M, Pan X, Han C, McPherson PS, Lacaille JC, Bonneil É, Thibault P, O'Leary C, Bigger B, Morales CR, Di Cristo G, and Pshezhetsky AV
- Subjects
- Animals, Behavior, Animal drug effects, Behavior, Animal physiology, Cells, Cultured, Cognitive Dysfunction drug therapy, Cognitive Dysfunction metabolism, Disease Progression, Drug Discovery, Hippocampus pathology, Mice, Neurodegenerative Diseases drug therapy, Neurodegenerative Diseases metabolism, Protein Transport, Lysosomal Storage Diseases metabolism, Mucopolysaccharidosis III metabolism, Mucopolysaccharidosis III psychology, Pyramidal Cells metabolism, Pyramidal Cells pathology, Secretory Vesicles metabolism, Synaptic Transmission physiology
- Abstract
The majority of patients affected with lysosomal storage disorders (LSD) exhibit neurological symptoms. For mucopolysaccharidosis type IIIC (MPSIIIC), the major burdens are progressive and severe neuropsychiatric problems and dementia, primarily thought to stem from neurodegeneration. Using the MPSIIIC mouse model, we studied whether clinical manifestations preceding massive neurodegeneration arise from synaptic dysfunction. Reduced levels or abnormal distribution of multiple synaptic proteins were revealed in cultured hippocampal and CA1 pyramidal MPSIIIC neurons. These defects were rescued by virus-mediated gene correction. Dendritic spines were reduced in pyramidal neurons of mouse models of MPSIIIC and other (Tay-Sachs, sialidosis) LSD as early as at P10. MPSIIIC neurons also presented alterations in frequency and amplitude of miniature excitatory and inhibitory postsynaptic currents, sparse synaptic vesicles, reduced postsynaptic densities, disorganized microtubule networks, and partially impaired axonal transport of synaptic proteins. Furthermore, postsynaptic densities were reduced in postmortem cortices of human MPS patients, suggesting that the pathology is a common hallmark for neurological LSD. Together, our results demonstrate that lysosomal storage defects cause early alterations in synaptic structure and abnormalities in neurotransmission originating from impaired synaptic vesicular transport, and they suggest that synaptic defects could be targeted to treat behavioral and cognitive defects in neurological LSD patients.
- Published
- 2021
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33. An Arf/Rab cascade controls the growth and invasiveness of glioblastoma.
- Author
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Kulasekaran G, Chaineau M, Piscopo VEC, Verginelli F, Fotouhi M, Girard M, Tang Y, Dali R, Lo R, Stifani S, and McPherson PS
- Subjects
- Allosteric Regulation, Animals, Cell Line, Tumor, Cell Movement, Cell Proliferation, Cell Self Renewal, ErbB Receptors metabolism, Guanine Nucleotide Exchange Factors chemistry, Guanine Nucleotide Exchange Factors metabolism, Humans, Male, Mice, Inbred NOD, Mice, SCID, Models, Biological, Neoplasm Invasiveness, Protein Binding, Protein Domains, Signal Transduction, Survival Analysis, Mice, ADP-Ribosylation Factors metabolism, Brain Neoplasms metabolism, Brain Neoplasms pathology, Glioblastoma metabolism, Glioblastoma pathology, rab GTP-Binding Proteins metabolism
- Abstract
Glioblastoma is the most common and deadly malignant brain cancer. We now demonstrate that loss of function of the endosomal GTPase Rab35 in human brain tumor initiating cells (BTICs) increases glioblastoma growth and decreases animal survival following BTIC implantation in mouse brains. Mechanistically, we identify that the GTPase Arf5 interacts with the guanine nucleotide exchange factor (GEF) for Rab35, DENND1/connecdenn, and allosterically enhances its GEF activity toward Rab35. Knockdown of either Rab35 or Arf5 increases cell migration, invasiveness, and self-renewal in culture and enhances the growth and invasiveness of BTIC-initiated brain tumors in mice. RNAseq of the tumors reveals up-regulation of the tumor-promoting transcription factor SPOCD1, and disruption of the Arf5/Rab35 axis in glioblastoma cells leads to strong activation of the epidermal growth factor receptor, with resulting enhancement of SPOCD1 levels. These discoveries reveal an unexpected cascade between an Arf and a Rab and indicate a role for the cascade, and thus endosomal trafficking, in brain tumors., (© 2021 Kulasekaran et al.)
- Published
- 2021
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34. SARS-CoV-2 infects cells after viral entry via clathrin-mediated endocytosis.
- Author
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Bayati A, Kumar R, Francis V, and McPherson PS
- Subjects
- A549 Cells, Angiotensin-Converting Enzyme 2 metabolism, Animals, Chlorocebus aethiops, Clathrin Heavy Chains antagonists & inhibitors, Clathrin Heavy Chains metabolism, Endocytosis drug effects, Endosomes drug effects, Endosomes metabolism, Endosomes virology, Gene Expression Regulation, Genetic Vectors chemistry, Genetic Vectors metabolism, HEK293 Cells, Host-Pathogen Interactions genetics, Humans, Hydrazones pharmacology, Lentivirus genetics, Lentivirus metabolism, Protein Binding drug effects, RNA, Small Interfering genetics, RNA, Small Interfering metabolism, SARS-CoV-2 drug effects, SARS-CoV-2 metabolism, Signal Transduction, Spike Glycoprotein, Coronavirus metabolism, Sulfonamides pharmacology, Thiazolidines pharmacology, Vero Cells, Angiotensin-Converting Enzyme 2 genetics, Clathrin Heavy Chains genetics, Endocytosis genetics, SARS-CoV-2 genetics, Spike Glycoprotein, Coronavirus genetics, Virus Internalization drug effects
- Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, so understanding its biology and infection mechanisms is critical to facing this major medical challenge. SARS-CoV-2 is known to use its spike glycoprotein to interact with the cell surface as a first step in the infection process. As for other coronaviruses, it is likely that SARS-CoV-2 next undergoes endocytosis, but whether or not this is required for infectivity and the precise endocytic mechanism used are unknown. Using purified spike glycoprotein and lentivirus pseudotyped with spike glycoprotein, a common model of SARS-CoV-2 infectivity, we now demonstrate that after engagement with the plasma membrane, SARS-CoV-2 undergoes rapid, clathrin-mediated endocytosis. This suggests that transfer of viral RNA to the cell cytosol occurs from the lumen of the endosomal system. Importantly, we further demonstrate that knockdown of clathrin heavy chain, which blocks clathrin-mediated endocytosis, reduces viral infectivity. These discoveries reveal that SARS-CoV-2 uses clathrin-mediated endocytosis to gain access into cells and suggests that this process is a key aspect of virus infectivity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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35. Regulated resurfacing of a somatostatin receptor storage compartment fine-tunes pituitary secretion.
- Author
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Alshafie W, Francis V, Bednarz K, Pan YE, Stroh T, and McPherson PS
- Subjects
- Animals, Corticotropin-Releasing Hormone, Humans, Mice, Inbred C57BL, Mice, Knockout, Pituitary Gland cytology, Qa-SNARE Proteins genetics, Receptors, Somatostatin genetics, Signal Transduction, Corticotrophs metabolism, Human Growth Hormone metabolism, Pituitary Gland metabolism, Qa-SNARE Proteins metabolism, Receptors, Somatostatin metabolism, rab GTP-Binding Proteins physiology
- Abstract
The surfacing of the glucose transporter GLUT4 driven by insulin receptor activation provides the prototypic example of a homeostasis response dependent on mobilization of an intracellular storage compartment. Here, we generalize this concept to a G protein-coupled receptor, somatostatin receptor subtype 2 (SSTR2), in pituitary cells. Following internalization in corticotropes, SSTR2 moves to a juxtanuclear syntaxin-6-positive compartment, where it remains until the corticotropes are stimulated with corticotropin releasing factor (CRF), whereupon SSTR2 exits the compartment on syntaxin-6-positive vesicular/tubular carriers that depend on Rab10 for their fusion with the plasma membrane. As SSTR2 activation antagonizes CRF-mediated hormone release, this storage/resurfacing mechanism may allow for a physiological homeostatic feedback system. In fact, we find that SSTR2 moves from an intracellular compartment to the cell surface in pituitary gland somatotropes, concomitant with increasing levels of serum growth hormone (GH) during natural GH cycles. Our data thus provide a mechanism by which signaling-mediated plasma membrane resurfacing of SSTR2 can fine-tune pituitary hormone release., (© 2019 McGill University.)
- Published
- 2020
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36. Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72.
- Author
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Laflamme C, McKeever PM, Kumar R, Schwartz J, Kolahdouzan M, Chen CX, You Z, Benaliouad F, Gileadi O, McBride HM, Durcan TM, Edwards AM, Healy LM, Robertson J, and McPherson PS
- Subjects
- Amyotrophic Lateral Sclerosis genetics, Amyotrophic Lateral Sclerosis immunology, Amyotrophic Lateral Sclerosis metabolism, Animals, Antibodies, Monoclonal classification, Antibodies, Monoclonal immunology, Biomarkers metabolism, C9orf72 Protein immunology, CRISPR-Cas Systems, Cell Line, Tumor, Frontotemporal Dementia genetics, Frontotemporal Dementia immunology, Frontotemporal Dementia metabolism, Gene Editing, Gene Expression, HEK293 Cells, Humans, Lysosomes genetics, Lysosomes metabolism, Lysosomes ultrastructure, Mice, Mice, Inbred C57BL, Mice, Transgenic, Osteoblasts metabolism, Osteoblasts ultrastructure, Phagosomes genetics, Phagosomes metabolism, Phagosomes ultrastructure, RAW 264.7 Cells, Amyotrophic Lateral Sclerosis diagnosis, Antibodies, Monoclonal chemistry, C9orf72 Protein genetics, Frontotemporal Dementia diagnosis, Immunohistochemistry standards
- Abstract
Antibodies are a key resource in biomedical research yet there are no community-accepted standards to rigorously characterize their quality. Here we develop a procedure to validate pre-existing antibodies. Human cell lines with high expression of a target, determined through a proteomics database, are modified with CRISPR/Cas9 to knockout (KO) the corresponding gene. Commercial antibodies against the target are purchased and tested by immunoblot comparing parental and KO. Validated antibodies are used to definitively identify the most highly expressing cell lines, new KOs are generated if needed, and the lines are screened by immunoprecipitation and immunofluorescence. Selected antibodies are used for more intensive procedures such as immunohistochemistry. The pipeline is easy to implement and scalable. Application to the major ALS disease gene C9ORF72 identified high-quality antibodies revealing C9ORF72 localization to phagosomes/lysosomes. Antibodies that do not recognize C9ORF72 have been used in highly cited papers, raising concern over previously reported C9ORF72 properties., Competing Interests: CL, PM, RK, JS, MK, CC, ZY, FB, OG, HM, TD, AE, LH, JR, PM No competing interests declared, (© 2019, Laflamme et al.)
- Published
- 2019
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37. SynGO: An Evidence-Based, Expert-Curated Knowledge Base for the Synapse.
- Author
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Koopmans F, van Nierop P, Andres-Alonso M, Byrnes A, Cijsouw T, Coba MP, Cornelisse LN, Farrell RJ, Goldschmidt HL, Howrigan DP, Hussain NK, Imig C, de Jong APH, Jung H, Kohansalnodehi M, Kramarz B, Lipstein N, Lovering RC, MacGillavry H, Mariano V, Mi H, Ninov M, Osumi-Sutherland D, Pielot R, Smalla KH, Tang H, Tashman K, Toonen RFG, Verpelli C, Reig-Viader R, Watanabe K, van Weering J, Achsel T, Ashrafi G, Asi N, Brown TC, De Camilli P, Feuermann M, Foulger RE, Gaudet P, Joglekar A, Kanellopoulos A, Malenka R, Nicoll RA, Pulido C, de Juan-Sanz J, Sheng M, Südhof TC, Tilgner HU, Bagni C, Bayés À, Biederer T, Brose N, Chua JJE, Dieterich DC, Gundelfinger ED, Hoogenraad C, Huganir RL, Jahn R, Kaeser PS, Kim E, Kreutz MR, McPherson PS, Neale BM, O'Connor V, Posthuma D, Ryan TA, Sala C, Feng G, Hyman SE, Thomas PD, Smit AB, and Verhage M
- Subjects
- Animals, Brain physiology, Databases, Genetic, Humans, Knowledge Bases, Synaptic Potentials physiology, Synaptosomes, Brain cytology, Gene Ontology, Proteomics, Software, Synapses physiology
- Abstract
Synapses are fundamental information-processing units of the brain, and synaptic dysregulation is central to many brain disorders ("synaptopathies"). However, systematic annotation of synaptic genes and ontology of synaptic processes are currently lacking. We established SynGO, an interactive knowledge base that accumulates available research about synapse biology using Gene Ontology (GO) annotations to novel ontology terms: 87 synaptic locations and 179 synaptic processes. SynGO annotations are exclusively based on published, expert-curated evidence. Using 2,922 annotations for 1,112 genes, we show that synaptic genes are exceptionally well conserved and less tolerant to mutations than other genes. Many SynGO terms are significantly overrepresented among gene variations associated with intelligence, educational attainment, ADHD, autism, and bipolar disorder and among de novo variants associated with neurodevelopmental disorders, including schizophrenia. SynGO is a public, universal reference for synapse research and an online analysis platform for interpretation of large-scale -omics data (https://syngoportal.org and http://geneontology.org)., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
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38. Macropinosome formation by tent pole ruffling in macrophages.
- Author
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Condon ND, Heddleston JM, Chew TL, Luo L, McPherson PS, Ioannou MS, Hodgson L, Stow JL, and Wall AA
- Subjects
- Actins genetics, Animals, CRISPR-Cas Systems, Cell Membrane Structures genetics, Gene Deletion, Lipopolysaccharides pharmacology, Mice, RAW 264.7 Cells, rab GTP-Binding Proteins genetics, Actins metabolism, Cell Membrane Structures metabolism, Macrophages metabolism, rab GTP-Binding Proteins metabolism
- Abstract
Pathogen-mediated activation of macrophages arms innate immune responses that include enhanced surface ruffling and macropinocytosis for environmental sampling and receptor internalization and signaling. Activation of macrophages with bacterial lipopolysaccharide (LPS) generates prominent dorsal ruffles, which are precursors for macropinosomes. Very rapid, high-resolution imaging of live macrophages with lattice light sheet microscopy (LLSM) reveals new features and actions of dorsal ruffles, which redefine the process of macropinosome formation and closure. We offer a new model in which ruffles are erected and supported by F-actin tent poles that cross over and twist to constrict the forming macropinosomes. This process allows for formation of large macropinosomes induced by LPS. We further describe the enrichment of active Rab13 on tent pole ruffles and show that CRISPR deletion of Rab13 results in aberrant tent pole ruffles and blocks the formation of large LPS-induced macropinosomes. Based on the exquisite temporal and spatial resolution of LLSM, we can redefine the ruffling and macropinosome processes that underpin innate immune responses., (© 2018 Condon et al.)
- Published
- 2018
- Full Text
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39. Structures of ubiquitin-like (Ubl) and Hsp90-like domains of sacsin provide insight into pathological mutations.
- Author
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Ménade M, Kozlov G, Trempe JF, Pande H, Shenker S, Wickremasinghe S, Li X, Hojjat H, Dicaire MJ, Brais B, McPherson PS, Wong MJH, Young JC, and Gehring K
- Subjects
- Amino Acid Substitution, Crystallography, X-Ray, Heat-Shock Proteins genetics, Heat-Shock Proteins metabolism, Humans, Muscle Spasticity genetics, Muscle Spasticity metabolism, Protein Domains, Spinocerebellar Ataxias congenital, Spinocerebellar Ataxias genetics, Spinocerebellar Ataxias metabolism, Heat-Shock Proteins chemistry, Mutation, Missense, Protein Folding
- Abstract
Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurodegenerative disease that is caused by mutations in the SACS gene. The product of this gene is a very large 520-kDa cytoplasmic protein, sacsin, with a ubiquitin-like (Ubl) domain at the N terminus followed by three large sacsin internal repeat (SIRPT) supradomains and C-terminal J and HEPN domains. The SIRPTs are predicted to contain Hsp90-like domains, suggesting a potential chaperone activity. In this work, we report the structures of the Hsp90-like Sr1 domain of SIRPT1 and the N-terminal Ubl domain determined at 1.55- and 2.1-Å resolutions, respectively. The Ubl domain crystallized as a swapped dimer that could be relevant in the context of full-length protein. The Sr1 domain displays the Bergerat protein fold with a characteristic nucleotide-binding pocket, although it binds nucleotides with very low affinity. The Sr1 structure reveals that ARSACS-causing missense mutations (R272H, R272C, and T201K) disrupt protein folding, most likely leading to sacsin degradation. This work lends structural support to the view of sacsin as a molecular chaperone and provides a framework for future studies of this protein., (© 2018 Ménade et al.)
- Published
- 2018
- Full Text
- View/download PDF
40. A PH-like domain of the Rab12 guanine nucleotide exchange factor DENND3 binds actin and is required for autophagy.
- Author
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Xu J, Kozlov G, McPherson PS, and Gehring K
- Subjects
- Actins chemistry, Actins genetics, Blood Proteins chemistry, Crystallography, X-Ray, Guanine Nucleotide Exchange Factors chemistry, Guanine Nucleotide Exchange Factors genetics, HEK293 Cells, Humans, Phosphoproteins chemistry, Phosphorylation, Protein Binding, Protein Conformation, Protein Domains, rab GTP-Binding Proteins chemistry, rab GTP-Binding Proteins genetics, Actins metabolism, Autophagy, Blood Proteins metabolism, Guanine Nucleotide Exchange Factors metabolism, Phosphoproteins metabolism, rab GTP-Binding Proteins metabolism
- Abstract
Rab GTPases are key regulators of membrane trafficking, and many are activated by guanine nucleotide exchange factors bearing a d ifferentially e xpressed in n ormal and n eoplastic cells (DENN) domain. By activating the small GTPase Rab12, DENN domain-containing protein 3 (DENND3) functions in autophagy. Here, we identified a structural domain (which we name PHenn) containing a pleckstrin homology subdomain that binds actin and is required for DENND3 function in autophagy. We found that a hydrophobic patch on an extended β-turn of the PHenn domain mediates an intramolecular interaction with the DENN domain of DENND3. We also show that DENND3 binds actin through a surface of positively charged residues on the PHenn domain. Substitutions that blocked either DENN or actin binding compromised the role of DENND3 in autophagy. These results provide new mechanistic insight into the structural determinants regulating DENND3 in autophagy and lay the foundation for future investigations of the DENN protein family., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2018
- Full Text
- View/download PDF
41. Light, space, and time in cancer signaling.
- Author
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McPherson PS and Wu M
- Subjects
- Congresses as Topic, Humans, Neoplasms genetics, Signal Transduction genetics
- Published
- 2018
- Full Text
- View/download PDF
42. Intersectin-s interaction with DENND2B facilitates recycling of epidermal growth factor receptor.
- Author
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Ioannou MS, Kulasekaran G, Fotouhi M, Morein JJ, Han C, Tse S, Nossova N, Han T, Mannard E, and McPherson PS
- Subjects
- Cell Membrane metabolism, Endocytosis, Epidermal Growth Factor pharmacology, ErbB Receptors drug effects, ErbB Receptors genetics, Guanine Nucleotide Exchange Factors genetics, HEK293 Cells, Humans, Neoplasms physiopathology, Phosphorylation, Protein Binding, Protein Kinase C metabolism, Protein Transport, Tumor Suppressor Proteins genetics, rab GTP-Binding Proteins metabolism, Adaptor Proteins, Vesicular Transport metabolism, Epidermal Growth Factor metabolism, ErbB Receptors metabolism, Guanine Nucleotide Exchange Factors metabolism, Signal Transduction, Tumor Suppressor Proteins metabolism
- Abstract
Epidermal growth factor (EGF) activates the EGF receptor (EGFR) and stimulates its internalization and trafficking to lysosomes for degradation. However, a percentage of EGFR undergoes ligand-independent endocytosis and is rapidly recycled back to the plasma membrane. Importantly, alterations in EGFR recycling are a common hallmark of cancer, and yet, our understanding of the machineries controlling the fate of endocytosed EGFR is incomplete. Intersectin-s is a multi-domain adaptor protein that is required for internalization of EGFR Here, we discover that intersectin-s binds DENND2B, a guanine nucleotide exchange factor for the exocytic GTPase Rab13, and this interaction promotes recycling of ligand-free EGFR to the cell surface. Intriguingly, upon EGF treatment, DENND2B is phosphorylated by protein kinase D and dissociates from intersectin-s, allowing for receptor targeting to degradation. Our study thus reveals a novel mechanism controlling the fate of internalized EGFR with important implications for cancer., (© 2017 The Authors.)
- Published
- 2017
- Full Text
- View/download PDF
43. Regulation of DENND3, the exchange factor for the small GTPase Rab12 through an intramolecular interaction.
- Author
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Xu J and McPherson PS
- Subjects
- Animals, Autophagy, Cell Membrane metabolism, Chromatography, Fluorescence Resonance Energy Transfer, Guanosine Diphosphate chemistry, Guanosine Triphosphate chemistry, HEK293 Cells, HeLa Cells, Humans, Immunoprecipitation, Mice, Microscopy, Fluorescence, Mutation, Phosphorylation, Protein Binding, Protein Domains, Tyrosine chemistry, Guanine Nucleotide Exchange Factors metabolism, rab GTP-Binding Proteins metabolism
- Abstract
The Rab family of small GTPases functions in multiple aspects of cellular membrane trafficking. Proteins bearing a d ifferentially e xpressed in n ormal and n eoplastic cells (DENN) domain have emerged as the largest family of Rab-activating guanine nucleotide exchange factors (GEFs). Rab12 functions in the initiation of starvation-induced autophagy, and our previous work revealed that its activator, DENN domain-containing protein 3 (DENND3), is phosphorylated and activated upon starvation. However, how the GEF activity of DENND3 toward Rab12 is regulated at the molecular level is still not understood. Here, we combine size-exclusion chromatography, Förster resonance energy transfer, pulldown, and in vitro GEF assays to demonstrate that regulation of GEF activity is achieved through an intramolecular interaction that is controlled by a key residue in DENND3, tyrosine 940. Our study sheds light on the regulation of Rab12 activation and lays the groundwork for characterizing the regulation of other DENN domain-containing proteins., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2017
- Full Text
- View/download PDF
44. Neurons Export Extracellular Vesicles Enriched in Cysteine String Protein and Misfolded Protein Cargo.
- Author
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Deng J, Koutras C, Donnelier J, Alshehri M, Fotouhi M, Girard M, Casha S, McPherson PS, Robbins SM, and Braun JEA
- Subjects
- Animals, Cells, Cultured, Humans, Mice, Neurons metabolism, Protein Folding, Synapses chemistry, Synapses metabolism, Extracellular Vesicles metabolism, HSP40 Heat-Shock Proteins metabolism, Membrane Proteins metabolism, Neurons cytology, Proteostasis
- Abstract
The fidelity of synaptic transmission depends on the integrity of the protein machinery at the synapse. Unfolded synaptic proteins undergo refolding or degradation in order to maintain synaptic proteostasis and preserve synaptic function, and buildup of unfolded/toxic proteins leads to neuronal dysfunction. Many molecular chaperones contribute to proteostasis, but one in particular, cysteine string protein (CSPα), is critical for proteostasis at the synapse. In this study we report that exported vesicles from neurons contain CSPα. Extracellular vesicles (EV's) have been implicated in a wide range of functions. However, the functional significance of neural EV's remains to be established. Here we demonstrate that co-expression of CSPα with the disease-associated proteins, polyglutamine expanded protein 72Q huntingtin
ex °n1 or superoxide dismutase-1 (SOD-1G93A) leads to the cellular export of both 72Q huntingtinex °n1 and SOD-1G93A via EV's. In contrast, the inactive CSPαHPD-AAA mutant does not facilitate elimination of misfolded proteins. Furthermore, CSPα-mediated export of 72Q huntingtinex °n1 is reduced by the polyphenol, resveratrol. Our results indicate that by assisting local lysosome/proteasome processes, CSPα-mediated removal of toxic proteins via EVs plays a central role in synaptic proteostasis and CSPα thus represents a potential therapeutic target for neurodegenerative diseases.- Published
- 2017
- Full Text
- View/download PDF
45. A lentiviral system for efficient knockdown of proteins in neuronal cultures [version 1; referees: 2 approved].
- Author
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Ritter B, Ferguson SM, De Camilli P, and McPherson PS
- Abstract
We have devised a protocol for highly efficient and specific knockdown of proteins in neuronal cultures. Small hairpin RNAs (shRNAs) are embedded into a microRNA (miRNA) context by oligo annealing to create shRNAmiRs, which are expressed from within the 3'-UTR of a reporter protein. This reporter protein/synthetic miRNA cassette is transferred to a targeting vector and lentivirus is produced in HEK-293-T cells following co-transfection of the targeting vector with three additional vectors encoding essential lentiviral proteins. Mature virus is harvested by collecting culture medium from transfected HEK-293-T cells, the virus is purified by centrifugation, and virus titers are determined prior to addition to neuronal cultures. Near 100% transduction efficiency of cultured hippocampal neurons is routinely observed and allows for the population-wide inhibition of target protein expression and the simultaneous knockdown of multiple proteins with little or no toxicity. The lentivirus generated can be used for protein knockdown in multiple neuronal culture models and at a variety of developmental stages. The steps from shRNAmiR design to ready-to-use virus stocks can be completed in as little as two weeks., Competing Interests: Competing interests: No competing interests were disclosed.
- Published
- 2017
- Full Text
- View/download PDF
46. Epileptic Encephalopathy Caused by Mutations in the Guanine Nucleotide Exchange Factor DENND5A.
- Author
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Han C, Alkhater R, Froukh T, Minassian AG, Galati M, Liu RH, Fotouhi M, Sommerfeld J, Alfrook AJ, Marshall C, Walker S, Bauer P, Scherer SW, Riess O, Buchert R, Minassian BA, and McPherson PS
- Subjects
- Adolescent, Animals, Child, Consanguinity, Female, Guanine Nucleotide Exchange Factors, Humans, Male, Neurons metabolism, PC12 Cells, Pedigree, Rats, Brain pathology, Epilepsy genetics, Mutation, rab GTP-Binding Proteins genetics
- Abstract
Epileptic encephalopathies are a catastrophic group of epilepsies characterized by refractory seizures and cognitive arrest, often resulting from abnormal brain development. Here, we have identified an epileptic encephalopathy additionally featuring cerebral calcifications and coarse facial features caused by recessive loss-of-function mutations in DENND5A. DENND5A contains a DENN domain, an evolutionarily ancient enzymatic module conferring guanine nucleotide exchange factor (GEF) activity to multiple proteins serving as GEFs for Rabs, which are key regulators of membrane trafficking. DENND5A is detected predominantly in neuronal tissues, and its highest levels occur during development. Knockdown of DENND5A leads to striking alterations in neuronal development. Mechanistically, these changes appear to result from upregulation of neurotrophin receptors, leading to enhanced downstream signaling. Thus, we have identified a link between a DENN domain protein and neuronal development, dysfunction of which is responsible for a form of epileptic encephalopathy., (Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
47. Rab13 Traffics on Vesicles Independent of Prenylation.
- Author
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Ioannou MS, Girard M, and McPherson PS
- Subjects
- Amino Acid Substitution, Animals, Cell Line, Cell Membrane Structures metabolism, Cytoplasmic Vesicles metabolism, Endosomes metabolism, Guanine Nucleotide Dissociation Inhibitors metabolism, HEK293 Cells, Humans, Models, Biological, Mutant Proteins chemistry, Mutant Proteins genetics, Mutant Proteins metabolism, Protein Interaction Domains and Motifs, Protein Prenylation, Protein Transport, Rats, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Single-Cell Analysis, rab GTP-Binding Proteins chemistry, rab GTP-Binding Proteins genetics, rab GTP-Binding Proteins metabolism
- Abstract
Rab GTPases are critical regulators of membrane trafficking. The canonical view is that Rabs are soluble in their inactive GDP-bound form, and only upon activation and conversion to their GTP-bound state are they anchored to membranes through membrane insertion of a C-terminal prenyl group. Here we demonstrate that C-terminal prenylation is not required for Rab13 to associate with and traffic on vesicles. Instead, inactive Rab13 appears to associate with vesicles via protein-protein interactions. Only following activation does Rab13 associate with the plasma membrane, presumably with insertion of the C-terminal prenyl group into the membrane., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
- Full Text
- View/download PDF
48. Rab-mediated membrane trafficking and the control of epithelial cell polarity.
- Author
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Ioannou MS and McPherson PS
- Subjects
- Epithelial Cells, Membranes, Cell Polarity, Protein Transport
- Abstract
Development of cell polarity requires apical trafficking of podocalyxin; yet the regulation of its transport is unclear. In this issue, Mrozowska and Fukuda (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201512024) demonstrate that different sets of Rabs and Rab effectors are used to regulate podocalyxin trafficking in two- versus three-dimensional model systems., (© 2016 Ioannou and McPherson.)
- Published
- 2016
- Full Text
- View/download PDF
49. Regulation of Cancer Cell Behavior by the Small GTPase Rab13.
- Author
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Ioannou MS and McPherson PS
- Subjects
- Animals, Biological Transport, Active, Cell Membrane genetics, Cell Membrane pathology, Humans, Neoplasm Proteins genetics, Neoplasms, rab GTP-Binding Proteins genetics, Cell Membrane metabolism, Cell Proliferation, Neoplasm Proteins metabolism, Signal Transduction, rab GTP-Binding Proteins metabolism
- Abstract
The members of the Rab family of GTPases are master regulators of cellular membrane trafficking. With ∼70 members in humans, Rabs have been implicated in all steps of membrane trafficking ranging from vesicle formation and transport to vesicle docking/tethering and fusion. Vesicle trafficking controls the localization and levels of a myriad of proteins, thus regulating cellular functions including proliferation, metabolism, cell-cell adhesion, and cell migration. It is therefore not surprising that impairment of Rab pathways is associated with diseases including cancer. In this review, we highlight evidence supporting the role of Rab13 as a potent driver of cancer progression., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
- Full Text
- View/download PDF
50. Endocytic membrane trafficking and neurodegenerative disease.
- Author
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Schreij AM, Fon EA, and McPherson PS
- Subjects
- Autophagy physiology, Clathrin-Coated Vesicles metabolism, Endocytosis genetics, Endocytosis physiology, Humans, Lysosomes metabolism, Protein Transport genetics, Amyotrophic Lateral Sclerosis genetics, Multivesicular Bodies metabolism, Parkinson Disease genetics, Protein Transport physiology, Spastic Paraplegia, Hereditary genetics
- Abstract
Neurodegenerative diseases are amongst the most devastating of human disorders. New technologies have led to a rapid increase in the identification of disease-related genes with an enhanced appreciation of the key roles played by genetics in the etiology of these disorders. Importantly, pinpointing the normal function of disease gene proteins leads to new understanding of the cellular machineries and pathways that are altered in the disease process. One such emerging pathway is membrane trafficking in the endosomal system. This key cellular process controls the localization and levels of a myriad of proteins and is thus critical for normal cell function. In this review we will focus on three neurodegenerative diseases; Parkinson disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegias, for which a large number of newly discovered disease genes encode proteins that function in endosomal membrane trafficking. We will describe how alterations in these proteins affect endosomal function and speculate on the contributions of these disruptions to disease pathophysiology.
- Published
- 2016
- Full Text
- View/download PDF
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