63 results on '"Fitzpatrick JAJ"'
Search Results
2. The centrosomal protein FGFR1OP controls myosin function in murine intestinal epithelial cells.
- Author
-
Trsan T, Peng V, Krishna C, Ohara TE, Beatty WL, Sudan R, Kanai M, Krishnamoorthy P, Rodrigues PF, Fachi JL, Grajales-Reyes G, Jaeger N, Fitzpatrick JAJ, Cella M, Gilfillan S, Nakata T, Jaiswal A, Stappenbeck TS, Daly MJ, Xavier RJ, and Colonna M
- Subjects
- Animals, Mice, Colitis metabolism, Colitis pathology, Colitis chemically induced, Colitis genetics, Centrosome metabolism, Humans, Cell Adhesion, Mice, Inbred C57BL, Crohn Disease metabolism, Crohn Disease pathology, Crohn Disease genetics, Actomyosin metabolism, Inflammation metabolism, Inflammation pathology, Inflammation genetics, Epithelial Cells metabolism, Intestinal Mucosa metabolism, Myosin Type II metabolism, Myosin Type II genetics
- Abstract
Recent advances in human genetics have shed light on the genetic factors contributing to inflammatory diseases, particularly Crohn's disease (CD), a prominent form of inflammatory bowel disease. Certain risk genes associated with CD directly influence cytokine biology and cell-specific communication networks. Current CD therapies primarily rely on anti-inflammatory drugs, which are inconsistently effective and lack strategies for promoting epithelial restoration and mucosal balance. To understand CD's underlying mechanisms, we investigated the link between CD and the FGFR1OP gene, which encodes a centrosome protein. FGFR1OP deletion in mouse intestinal epithelial cells disrupted crypt architecture, resulting in crypt loss, inflammation, and fatality. FGFR1OP insufficiency hindered epithelial resilience during colitis. FGFR1OP was crucial for preserving non-muscle myosin II activity, ensuring the integrity of the actomyosin cytoskeleton and crypt cell adhesion. This role of FGFR1OP suggests that its deficiency in genetically predisposed individuals may reduce epithelial renewal capacity, heightening susceptibility to inflammation and disease., Competing Interests: Declaration of interests R.J.X. is co-founder of Jnana Therapeutics and Celsius Therapeutics, scientific advisory board member at Nestlé, and board director at MoonLake Immunotherapeutics. These organizations had no roles in this study., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
- Full Text
- View/download PDF
3. Signaling by a bacterial phytochrome histidine kinase involves a conformational cascade reorganizing the dimeric photoreceptor.
- Author
-
Burgie ES, Basore K, Rau MJ, Summers B, Mickles AJ, Grigura V, Fitzpatrick JAJ, and Vierstra RD
- Subjects
- Models, Molecular, Cryoelectron Microscopy, Protein Conformation, Protein Multimerization, Photoreceptors, Microbial metabolism, Photoreceptors, Microbial chemistry, Photoreceptors, Microbial genetics, Light, Histidine Kinase metabolism, Histidine Kinase chemistry, Histidine Kinase genetics, Bacterial Proteins metabolism, Bacterial Proteins chemistry, Bacterial Proteins genetics, Phytochrome metabolism, Phytochrome chemistry, Signal Transduction, Pseudomonas syringae metabolism
- Abstract
Phytochromes (Phys) are a divergent cohort of bili-proteins that detect light through reversible interconversion between dark-adapted Pr and photoactivated Pfr states. While our understandings of downstream events are emerging, it remains unclear how Phys translate light into an interpretable conformational signal. Here, we present models of both states for a dimeric Phy with histidine kinase (HK) activity from the proteobacterium Pseudomonas syringae, which were built from high-resolution cryo-EM maps (2.8-3.4-Å) of the photosensory module (PSM) and its following signaling (S) helix together with lower resolution maps for the downstream output region augmented by RoseTTAFold and AlphaFold structural predictions. The head-to-head models reveal the PSM and its photointerconversion mechanism with strong clarity, while the HK region is interpretable but relatively mobile. Pr/Pfr comparisons show that bilin phototransformation alters PSM architecture culminating in a scissoring motion of the paired S-helices linking the PSMs to the HK bidomains that ends in reorientation of the paired catalytic ATPase modules relative to the phosphoacceptor histidines. This action apparently primes autophosphorylation enroute to phosphotransfer to the cognate DNA-binding response regulator AlgB which drives quorum-sensing behavior through transient association with the photoreceptor. Collectively, these models illustrate how light absorption conformationally translates into accelerated signaling by Phy-type kinases., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
4. Modeling late-onset Alzheimer's disease neuropathology via direct neuronal reprogramming.
- Author
-
Sun Z, Kwon JS, Ren Y, Chen S, Walker CK, Lu X, Cates K, Karahan H, Sviben S, Fitzpatrick JAJ, Valdez C, Houlden H, Karch CM, Bateman RJ, Sato C, Mennerick SJ, Diamond MI, Kim J, Tanzi RE, Holtzman DM, and Yoo AS
- Subjects
- Humans, Amyloid Precursor Protein Secretases antagonists & inhibitors, Amyloid Precursor Protein Secretases metabolism, Amyloid Precursor Protein Secretases genetics, Alzheimer Disease pathology, Alzheimer Disease genetics, Alzheimer Disease metabolism, Amyloid beta-Peptides metabolism, Cellular Reprogramming genetics, Fibroblasts metabolism, Fibroblasts pathology, MicroRNAs genetics, MicroRNAs metabolism, Neurons metabolism, Neurons pathology, Spheroids, Cellular
- Abstract
Late-onset Alzheimer's disease (LOAD) is the most common form of Alzheimer's disease (AD). However, modeling sporadic LOAD that endogenously captures hallmark neuronal pathologies such as amyloid-β (Aβ) deposition, tau tangles, and neuronal loss remains an unmet need. We demonstrate that neurons generated by microRNA (miRNA)-based direct reprogramming of fibroblasts from individuals affected by autosomal dominant AD (ADAD) and LOAD in a three-dimensional environment effectively recapitulate key neuropathological features of AD. Reprogrammed LOAD neurons exhibit Aβ-dependent neurodegeneration, and treatment with β- or γ-secretase inhibitors before (but not subsequent to) Aβ deposit formation mitigated neuronal death. Moreover inhibiting age-associated retrotransposable elements in LOAD neurons reduced both Aβ deposition and neurodegeneration. Our study underscores the efficacy of modeling late-onset neuropathology of LOAD through high-efficiency miRNA-based neuronal reprogramming.
- Published
- 2024
- Full Text
- View/download PDF
5. Poly-basic peptides and polymers as new drug candidates against Plasmodium falciparum.
- Author
-
Sivakumar R, Floyd K, Erath J, Jacoby A, Kim Kim J, Bayguinov PO, Fitzpatrick JAJ, Goldfarb D, Jovanovic M, Tripathi A, Djuranovic S, and Pavlovic-Djuranovic S
- Subjects
- Peptides pharmacology, Peptides chemistry, Humans, Polymers pharmacology, Polymers chemistry, Polylysine pharmacology, Polylysine chemistry, Plasmodium falciparum drug effects, Antimalarials pharmacology, Antimalarials chemistry, Erythrocytes drug effects, Erythrocytes parasitology
- Abstract
Background: Plasmodium falciparum, the malaria-causing parasite, is a leading cause of infection-induced deaths worldwide. The preferred treatment approach is artemisinin-based combination therapy, which couples fast-acting artemisinin derivatives with longer-acting drugs, such as lumefantrine, mefloquine, and amodiaquine. However, the urgency for new treatments has risen due to the parasite's growing resistance to existing therapies. In this study, a common characteristic of the P. falciparum proteome-stretches of poly-lysine residues, such as those found in proteins related to adhesion and pathogenicity-is investigated for its potential to treat infected erythrocytes., Methods: This study utilizes in vitro culturing of intra-erythrocytic P. falciparum to assess the ability of poly-lysine peptides to inhibit the parasite's growth, measured via flow cytometry of acridine orange-stained infected erythrocytes. The inhibitory effect of many poly-lysine lengths and modifications were tested this way. Affinity pull-downs and mass spectrometry were performed to identify the proteins interacting with these poly-lysines., Results: A single dose of these poly-basic peptides can successfully diminish parasitemia in human erythrocytes in vitro with minimal toxicity. The effectiveness of the treatment correlates with the length of the poly-lysine peptide, with 30 lysine peptides supporting the eradication of erythrocytic parasites within 72 h. PEG-ylation of the poly-lysine peptides or utilizing poly-lysine dendrimers and polymers retains or increases parasite clearance efficiency and bolsters the stability of these potential new therapeutics. Lastly, affinity pull-downs and mass-spectrometry identify P. falciparum's outer membrane proteins as likely targets for polybasic peptide medications., Conclusion: Since poly-lysine dendrimers are already FDA-approved for drug delivery and this study displays their potency against intraerythrocytic P. falciparum, their adaptation as anti-malarial drugs presents a promising new therapeutic strategy for malaria., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
6. Apolipoprotein E secreted by astrocytes forms antiparallel dimers in discoidal lipoproteins.
- Author
-
Strickland MR, Rau MJ, Summers B, Basore K, Wulf J 2nd, Jiang H, Chen Y, Ulrich JD, Randolph GJ, Zhang R, Fitzpatrick JAJ, Cashikar AG, and Holtzman DM
- Subjects
- Lipoproteins, HDL chemistry, Lipoproteins, HDL metabolism, Central Nervous System metabolism, Apolipoprotein E4 metabolism, Apolipoprotein E3 metabolism, Astrocytes metabolism, Apolipoproteins E genetics, Lipoproteins
- Abstract
The Apolipoprotein E gene (APOE) is of great interest due to its role as a risk factor for late-onset Alzheimer's disease. ApoE is secreted by astrocytes in the central nervous system in high-density lipoprotein (HDL)-like lipoproteins. Structural models of lipidated ApoE of high resolution could aid in a mechanistic understanding of how ApoE functions in health and disease. Using monoclonal Fab and F(ab')
2 fragments, we characterize the structure of lipidated ApoE on astrocyte-secreted lipoproteins. Our results provide support for the "double-belt" model of ApoE in nascent discoidal HDL-like lipoproteins, where two ApoE proteins wrap around the nanodisc in an antiparallel conformation. We further show that lipidated, recombinant ApoE accurately models astrocyte-secreted ApoE lipoproteins. Cryogenic electron microscopy of recombinant lipidated ApoE further supports ApoE adopting antiparallel dimers in nascent discoidal lipoproteins., Competing Interests: Declaration of interests D.M.H. is an inventor on a patent licensed by Washington University to NextCure on the therapeutic use of anti-ApoE antibodies. D.M.H. co-founded and is on the scientific advisory board of C2N Diagnostics. D.M.H. is on the scientific advisory board of Denali, Genentech, and Cajal Neuroscience; consults for Asteroid; and is on the advisory board for Cell., (Copyright © 2024 Elsevier Inc. All rights reserved.)- Published
- 2024
- Full Text
- View/download PDF
7. Structure of alpha-synuclein fibrils derived from human Lewy body dementia tissue.
- Author
-
Dhavale DD, Barclay AM, Borcik CG, Basore K, Berthold DA, Gordon IR, Liu J, Milchberg MH, O'Shea JY, Rau MJ, Smith Z, Sen S, Summers B, Smith J, Warmuth OA, Perrin RJ, Perlmutter JS, Chen Q, Fitzpatrick JAJ, Schwieters CD, Tajkhorshid E, Rienstra CM, and Kotzbauer PT
- Subjects
- Humans, alpha-Synuclein chemistry, Cryoelectron Microscopy, Lewy Bodies pathology, Lewy Body Disease pathology, Parkinson Disease pathology
- Abstract
The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. Here we develop and validate a method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and use solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. Amplified LBD Asyn fibrils comprise a mixture of single protofilament and two protofilament fibrils with very low twist. The protofilament fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural characterization of LBD Asyn fibrils and approaches for studying disease mechanisms, imaging agents and therapeutics targeting Asyn., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
8. Lipid nanodisc scaffold and size alter the structure of a pentameric ligand-gated ion channel.
- Author
-
Dalal V, Arcario MJ, Petroff JT 2nd, Tan BK, Dietzen NM, Rau MJ, Fitzpatrick JAJ, Brannigan G, and Cheng WWL
- Subjects
- Cryoelectron Microscopy, Models, Molecular, Binding Sites, Lipids, Ligand-Gated Ion Channels chemistry, Ligand-Gated Ion Channels metabolism
- Abstract
Lipid nanodiscs have become a standard tool for studying membrane proteins, including using single particle cryo-electron microscopy (cryo-EM). We find that reconstituting the pentameric ligand-gated ion channel (pLGIC), Erwinia ligand-gated ion channel (ELIC), in different nanodiscs produces distinct structures by cryo-EM. The effect of the nanodisc on ELIC structure extends to the extracellular domain and agonist binding site. Additionally, molecular dynamic simulations indicate that nanodiscs of different size impact ELIC structure and that the nanodisc scaffold directly interacts with ELIC. These findings suggest that the nanodisc plays a crucial role in determining the structure of pLGICs, and that reconstitution of ion channels in larger nanodiscs may better approximate a lipid membrane environment., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
9. Multiple Genes Core to ERAD, Macroautophagy and Lysosomal Degradation Pathways Participate in the Proteostasis Response in α1-Antitrypsin Deficiency.
- Author
-
Li J, Moretti F, Hidvegi T, Sviben S, Fitzpatrick JAJ, Sundaramoorthi H, Pak SC, Silverman GA, Knapp B, Filipuzzi I, Alford J, Reece-Hoyes J, Nigsch F, Murphy LO, Nyfeler B, and Perlmutter DH
- Subjects
- Humans, CRISPR-Cas Systems, Autophagy genetics, Gene Editing, alpha 1-Antitrypsin Deficiency pathology, alpha 1-Antitrypsin Deficiency genetics, alpha 1-Antitrypsin Deficiency metabolism, Proteostasis, Lysosomes metabolism, alpha 1-Antitrypsin metabolism, alpha 1-Antitrypsin genetics, Endoplasmic Reticulum-Associated Degradation, Macroautophagy, Endoplasmic Reticulum metabolism
- Abstract
Background & Aims: In the classic form of α1-antitrypsin deficiency (ATD), the misfolded α1-antitrypsin Z (ATZ) variant accumulates in the endoplasmic reticulum (ER) of liver cells. A gain-of-function proteotoxic mechanism is responsible for chronic liver disease in a subgroup of homozygotes. Proteostatic response pathways, including conventional endoplasmic reticulum-associated degradation and autophagy, have been proposed as the mechanisms that allow cellular adaptation and presumably protection from the liver disease phenotype. Recent studies have concluded that a distinct lysosomal pathway called endoplasmic reticulum-to-lysosome completely supplants the role of the conventional macroautophagy pathway in degradation of ATZ. Here, we used several state-of-the-art approaches to characterize the proteostatic responses more fully in cellular systems that model ATD., Methods: We used clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing coupled to a cell selection step by fluorescence-activated cell sorter to perform screening for proteostasis genes that regulate ATZ accumulation and combined that with selective genome editing in 2 other model systems., Results: Endoplasmic reticulum-associated degradation genes are key early regulators and multiple autophagy genes, from classic as well as from ER-to-lysosome and other newly described ER-phagy pathways, participate in degradation of ATZ in a manner that is temporally regulated and evolves as ATZ accumulation persists. Time-dependent changes in gene expression are accompanied by specific ultrastructural changes including dilation of the ER, formation of globular inclusions, budding of autophagic vesicles, and alterations in the overall shape and component parts of mitochondria., Conclusions: Macroautophagy is a critical component of the proteostasis response to cellular ATZ accumulation and it becomes more important over time as ATZ synthesis continues unabated. Multiple subtypes of macroautophagy and nonautophagic lysosomal degradative pathways are needed to respond to the high concentrations of misfolded protein that characterizes ATD and these pathways are attractive candidates for genetic variants that predispose to the hepatic phenotype., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
- Full Text
- View/download PDF
10. Yeast Rad52 is a homodecamer and possesses BRCA2-like bipartite Rad51 binding modes.
- Author
-
Deveryshetty J, Chadda R, Mattice JR, Karunakaran S, Rau MJ, Basore K, Pokhrel N, Englander N, Fitzpatrick JAJ, Bothner B, and Antony E
- Subjects
- Cryoelectron Microscopy, DNA Repair, DNA, Single-Stranded metabolism, Protein Binding, Rad51 Recombinase metabolism, Rad52 DNA Repair and Recombination Protein genetics, Rad52 DNA Repair and Recombination Protein metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Homologous recombination (HR) is an essential double-stranded DNA break repair pathway. In HR, Rad52 facilitates the formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Here, we decipher how Rad52 functions using single-particle cryo-electron microscopy and biophysical approaches. We report that Rad52 is a homodecameric ring and each subunit possesses an ordered N-terminal and disordered C-terminal half. An intrinsic structural asymmetry is observed where a few of the C-terminal halves interact with the ordered ring. We describe two conserved charged patches in the C-terminal half that harbor Rad51 and RPA interacting motifs. Interactions between these patches regulate ssDNA binding. Surprisingly, Rad51 interacts with Rad52 at two different bindings sites: one within the positive patch in the disordered C-terminus and the other in the ordered ring. We propose that these features drive Rad51 nucleation onto a single position on the DNA to promote formation of uniform pre-synaptic Rad51 filaments in HR., (© 2023. Springer Nature Limited.)
- Published
- 2023
- Full Text
- View/download PDF
11. Poly-basic peptides and polymers as new drug candidate against Plasmodium falciparum .
- Author
-
Sivakumar R, Floyd K, Jessey E, Kim JK, Bayguinov PO, Fitzpatrick JAJ, Goldfrab D, Jovanovic M, Tripathi A, Djuranovic S, and Pavlovic-Djuranovic S
- Abstract
Plasmodium falciparum , the malaria-causing parasite, is a leading cause of infection-induced deaths worldwide. The preferred treatment approach is artemisinin-combination therapy, which couples fast-acting artemisinin derivatives with longer-acting drugs like lumefantrine, mefloquine, and amodiaquine. However, the urgency for new treatments has risen due to the parasite's growing resistance to existing therapies. Our study shows that a common characteristic of the P. falciparum proteome - stretches of poly-lysine residues such as those found in proteins related to adhesion and pathogenicity - can serve as an effective peptide treatment for infected erythrocytes. A single dose of these poly-basic peptides can successfully diminish parasitemia in human erythrocytes in vitro with minimal toxicity. The effectiveness of the treatment correlates with the length of the poly-lysine peptide, with 30 lysine peptides supporting the eradication of erythrocytic parasites within 72 hours. PEG-ylation of the poly-lysine peptides or utilizing poly-lysine dendrimers and polymers further increases parasite clearance efficiency and bolsters the stability of these potential new therapeutics. Lastly, our affinity pull-downs and mass-spectrometry identify P. falciparum's outer membrane proteins as likely targets for polybasic peptide medications. Since poly-lysine dendrimers are already FDA-approved for drug delivery, their adaptation as antimalarial drugs presents a promising new therapeutic strategy., Competing Interests: Competing interests: Authors do not have competing interests. Authors SD, SPD, and JE hold US Provisional Patent Application Serial No. 62/696,868 “Antimalarial Compositions and Methods of Use.” Associated with this study.
- Published
- 2023
- Full Text
- View/download PDF
12. Deletion of taf1 and taf5 in zebrafish capitulate cardiac and craniofacial abnormalities associated with TAFopathies through perturbations in metabolism.
- Author
-
Leid J, Gray R, Rakita P, Koenig AL, Tripathy R, Fitzpatrick JAJ, Kaufman C, Solnica-Krezel L, and Lavine KJ
- Subjects
- Animals, Heart, Intellectual Disability, Mutation, Transcription Factor TFIID genetics, Zebrafish, Craniofacial Abnormalities genetics, TATA-Binding Protein Associated Factors genetics, Zebrafish Proteins genetics
- Abstract
Intellectual disability is a neurodevelopmental disorder that affects 2-3% of the general population. Syndromic forms of intellectual disability frequently have a genetic basis and are often accompanied by additional developmental anomalies. Pathogenic variants in components of TATA-binding protein associated factors (TAFs) have recently been identified in a subset of patients with intellectual disability, craniofacial hypoplasia, and congenital heart disease. This syndrome has been termed as a TAFopathy and includes mutations in TATA binding protein (TBP), TAF1, TAF2, and TAF6. The underlying mechanism by which TAFopathies give rise to neurodevelopmental, craniofacial, and cardiac abnormalities remains to be defined. Through a forward genetic screen in zebrafish, we have recovered a recessive mutant phenotype characterized by craniofacial hypoplasia, ventricular hypoplasia, heart failure at 96 h post-fertilization and lethality, and show it is caused by a nonsense mutation in taf5. CRISPR/CAS9 mediated gene editing revealed that these defects where phenocopied by mutations in taf1 and taf5. Mechanistically, taf5-/- zebrafish displayed misregulation in metabolic gene expression and metabolism as evidenced by RNA sequencing, respiration assays, and metabolite studies. Collectively, these findings suggest that the TAF complex may contribute to neurologic, craniofacial, and cardiac development through regulation of metabolism., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)
- Published
- 2023
- Full Text
- View/download PDF
13. Homodecameric Rad52 promotes single-position Rad51 nucleation in homologous recombination.
- Author
-
Deveryshetty J, Chadda R, Mattice J, Karunakaran S, Rau MJ, Basore K, Pokhrel N, Englander N, Fitzpatrick JAJ, Bothner B, and Antony E
- Abstract
Homologous recombination (HR) is a pathway for the accurate repair of double-stranded DNA breaks. These breaks are resected to yield single-stranded DNA (ssDNA) that are coated by Replication Protein A (RPA). Saccharomyces cerevisiae Rad52 is a mediator protein that promotes HR by facilitating formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Canonically, Rad52 has been described to function by displacing RPA to promote Rad51 binding. However, in vitro , Rad51 readily forms a filament by displacing RPA in the absence of Rad52. Yet, in vivo , Rad52 is essential for HR. Here, we resolve how Rad52 functions as a mediator using single-particle cryo-electron microscopy and biophysical approaches. We show that Rad52 functions as a homodecamer and catalyzes single-position nucleation of Rad51. The N-terminal half of Rad52 is a well-ordered ring, while the C-terminal half is disordered. An intrinsic asymmetry within Rad52 is observed, where one or a few of the C-terminal halves interact with the ordered N-terminal ring. Within the C-terminal half, we identify two conserved charged patches that harbor the Rad51 and RPA interacting motifs. Interactions between these two charged patches regulate a ssDNA binding. These features drive Rad51 binding to a single position on the Rad52 decameric ring. We propose a Rad52 catalyzed single-position nucleation model for the formation of pre-synaptic Rad51 filaments in HR., Competing Interests: Competing interests The authors declare no competing interests.
- Published
- 2023
- Full Text
- View/download PDF
14. Endogenous recapitulation of Alzheimer's disease neuropathology through human 3D direct neuronal reprogramming.
- Author
-
Sun Z, Kwon JS, Ren Y, Chen S, Cates K, Lu X, Walker CK, Karahan H, Sviben S, Fitzpatrick JAJ, Valdez C, Houlden H, Karch CM, Bateman RJ, Sato C, Mennerick SJ, Diamond MI, Kim J, Tanzi RE, Holtzman DM, and Yoo AS
- Abstract
Alzheimer's disease (AD) is a neurodegenerative disorder that primarily affects elderly individuals, and is characterized by hallmark neuronal pathologies including extracellular amyloid-β (Aβ) plaque deposition, intracellular tau tangles, and neuronal death. However, recapitulating these age-associated neuronal pathologies in patient-derived neurons has remained a significant challenge, especially for late-onset AD (LOAD), the most common form of the disorder. Here, we applied the high efficiency microRNA-mediated direct neuronal reprogramming of fibroblasts from AD patients to generate cortical neurons in three-dimensional (3D) Matrigel and self-assembled neuronal spheroids. Our findings indicate that neurons and spheroids reprogrammed from both autosomal dominant AD (ADAD) and LOAD patients exhibited AD-like phenotypes linked to neurons, including extracellular Aβ deposition, dystrophic neurites with hyperphosphorylated, K63-ubiquitin-positive, seed-competent tau, and spontaneous neuronal death in culture. Moreover, treatment with β- or γ-secretase inhibitors in LOAD patient-derived neurons and spheroids before Aβ deposit formation significantly lowered Aβ deposition, as well as tauopathy and neurodegeneration. However, the same treatment after the cells already formed Aβ deposits only had a mild effect. Additionally, inhibiting the synthesis of age-associated retrotransposable elements (RTEs) by treating LOAD neurons and spheroids with the reverse transcriptase inhibitor, lamivudine, alleviated AD neuropathology. Overall, our results demonstrate that direct neuronal reprogramming of AD patient fibroblasts in a 3D environment can capture age-related neuropathology and reflect the interplay between Aβ accumulation, tau dysregulation, and neuronal death. Moreover, miRNA-based 3D neuronal conversion provides a human-relevant AD model that can be used to identify compounds that can potentially ameliorate AD-associated pathologies and neurodegeneration.
- Published
- 2023
- Full Text
- View/download PDF
15. Transcriptomic atlas and interaction networks of brain cells in mouse CNS demyelination and remyelination.
- Author
-
Hou J, Zhou Y, Cai Z, Terekhova M, Swain A, Andhey PS, Guimaraes RM, Ulezko Antonova A, Qiu T, Sviben S, Strout G, Fitzpatrick JAJ, Chen Y, Gilfillan S, Kim DH, Van Dyken SJ, Artyomov MN, and Colonna M
- Subjects
- Animals, Mice, Transcriptome genetics, Brain metabolism, Oligodendroglia metabolism, Microglia metabolism, Cuprizone toxicity, Disease Models, Animal, Mice, Inbred C57BL, Myelin Sheath metabolism, Demyelinating Diseases metabolism, Remyelination
- Abstract
Demyelination is a hallmark of multiple sclerosis, leukoencephalopathies, cerebral vasculopathies, and several neurodegenerative diseases. The cuprizone mouse model is widely used to simulate demyelination and remyelination occurring in these diseases. Here, we present a high-resolution single-nucleus RNA sequencing (snRNA-seq) analysis of gene expression changes across all brain cells in this model. We define demyelination-associated oligodendrocytes (DOLs) and remyelination-associated MAFB
hi microglia, as well as astrocytes and vascular cells with signatures of altered metabolism, oxidative stress, and interferon response. Furthermore, snRNA-seq provides insights into how brain cell types connect and interact, defining complex circuitries that impact demyelination and remyelination. As an explicative example, perturbation of microglia caused by TREM2 deficiency indirectly impairs the induction of DOLs. Altogether, this study provides a rich resource for future studies investigating mechanisms underlying demyelinating diseases., Competing Interests: Declaration of interests M.C. is a member of the scientific advisory board of Vigil, receives research support from Vigil, and is a consultant for CST., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2023
- Full Text
- View/download PDF
16. Structural and functional analysis of human pannexin 2 channel.
- Author
-
He Z, Zhao Y, Rau MJ, Fitzpatrick JAJ, Sah R, Hu H, and Yuan P
- Subjects
- Humans, Anions, Biological Transport, Cryoelectron Microscopy, Connexins metabolism, Membrane Proteins metabolism, Nerve Tissue Proteins genetics, Nerve Tissue Proteins metabolism
- Abstract
The pannexin 2 channel (PANX2) participates in multiple physiological processes including skin homeostasis, neuronal development, and ischemia-induced brain injury. However, the molecular basis of PANX2 channel function remains largely unknown. Here, we present a cryo-electron microscopy structure of human PANX2, which reveals pore properties contrasting with those of the intensely studied paralog PANX1. The extracellular selectivity filter, defined by a ring of basic residues, more closely resembles that of the distantly related volume-regulated anion channel (VRAC) LRRC8A, rather than PANX1. Furthermore, we show that PANX2 displays a similar anion permeability sequence as VRAC, and that PANX2 channel activity is inhibited by a commonly used VRAC inhibitor, DCPIB. Thus, the shared channel properties between PANX2 and VRAC may complicate dissection of their cellular functions through pharmacological manipulation. Collectively, our structural and functional analysis provides a framework for development of PANX2-specific reagents that are needed for better understanding of channel physiology and pathophysiology., (© 2023. The Author(s).)
- Published
- 2023
- Full Text
- View/download PDF
17. Gold nanoparticle-enhanced X-ray microtomography of the rodent reveals region-specific cerebrospinal fluid circulation in the brain.
- Author
-
Pan S, Yang PH, DeFreitas D, Ramagiri S, Bayguinov PO, Hacker CD, Snyder AZ, Wilborn J, Huang H, Koller GM, Raval DK, Halupnik GL, Sviben S, Achilefu S, Tang R, Haller G, Quirk JD, Fitzpatrick JAJ, Esakky P, and Strahle JM
- Subjects
- Animals, Gold metabolism, Rodentia, X-Ray Microtomography, Brain metabolism, Cerebrospinal Fluid metabolism, Metal Nanoparticles, Hydrocephalus
- Abstract
Cerebrospinal fluid (CSF) is essential for the development and function of the central nervous system (CNS). However, the brain and its interstitium have largely been thought of as a single entity through which CSF circulates, and it is not known whether specific cell populations within the CNS preferentially interact with the CSF. Here, we develop a technique for CSF tracking, gold nanoparticle-enhanced X-ray microtomography, to achieve micrometer-scale resolution visualization of CSF circulation patterns during development. Using this method and subsequent histological analysis in rodents, we identify previously uncharacterized CSF pathways from the subarachnoid space (particularly the basal cisterns) that mediate CSF-parenchymal interactions involving 24 functional-anatomic cell groupings in the brain and spinal cord. CSF distribution to these areas is largely restricted to early development and is altered in posthemorrhagic hydrocephalus. Our study also presents particle size-dependent CSF circulation patterns through the CNS including interaction between neurons and small CSF tracers, but not large CSF tracers. These findings have implications for understanding the biological basis of normal brain development and the pathogenesis of a broad range of disease states, including hydrocephalus., (© 2023. The Author(s).)
- Published
- 2023
- Full Text
- View/download PDF
18. Structure of alpha-synuclein fibrils derived from human Lewy body dementia tissue.
- Author
-
Dhavale DD, Barclay AM, Borcik CG, Basore K, Gordon IR, Liu J, Milchberg MH, O'shea J, Rau MJ, Smith Z, Sen S, Summers B, Smith J, Warmuth OA, Chen Q, Fitzpatrick JAJ, Schwieters CD, Tajkhorshid E, Rienstra CM, and Kotzbauer PT
- Abstract
The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. We developed and validated a novel method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and used solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. Amplified LBD Asyn fibrils comprise two protofilaments with pseudo-2
1 helical screw symmetry, very low twist and an interface formed by antiparallel beta strands of residues 85-93. The fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural landscape of LBD Asyn fibrils and inform further studies of disease mechanisms, imaging agents and therapeutics targeting Asyn.- Published
- 2023
- Full Text
- View/download PDF
19. Open-channel structure of a pentameric ligand-gated ion channel reveals a mechanism of leaflet-specific phospholipid modulation.
- Author
-
Petroff JT 2nd, Dietzen NM, Santiago-McRae E, Deng B, Washington MS, Chen LJ, Trent Moreland K, Deng Z, Rau M, Fitzpatrick JAJ, Yuan P, Joseph TT, Hénin J, Brannigan G, and Cheng WWL
- Subjects
- Phospholipids, Binding Sites, Phosphatidylglycerols, Liposomes, Ligand-Gated Ion Channels chemistry, Ligand-Gated Ion Channels metabolism
- Abstract
Pentameric ligand-gated ion channels (pLGICs) mediate synaptic transmission and are sensitive to their lipid environment. The mechanism of phospholipid modulation of any pLGIC is not well understood. We demonstrate that the model pLGIC, ELIC (Erwinia ligand-gated ion channel), is positively modulated by the anionic phospholipid, phosphatidylglycerol, from the outer leaflet of the membrane. To explore the mechanism of phosphatidylglycerol modulation, we determine a structure of ELIC in an open-channel conformation. The structure shows a bound phospholipid in an outer leaflet site, and structural changes in the phospholipid binding site unique to the open-channel. In combination with streamlined alchemical free energy perturbation calculations and functional measurements in asymmetric liposomes, the data support a mechanism by which an anionic phospholipid stabilizes the activated, open-channel state of a pLGIC by specific, state-dependent binding to this site., (© 2022. The Author(s).)
- Published
- 2022
- Full Text
- View/download PDF
20. Structural basis for mechanotransduction in a potassium-dependent mechanosensitive ion channel.
- Author
-
Mount J, Maksaev G, Summers BT, Fitzpatrick JAJ, and Yuan P
- Subjects
- Mechanotransduction, Cellular, Cryoelectron Microscopy, Models, Molecular, Ion Channels metabolism, Potassium, Potassium Channels metabolism, Ion Channel Gating
- Abstract
Mechanosensitive channels of small conductance, found in many living organisms, open under elevated membrane tension and thus play crucial roles in biological response to mechanical stress. Amongst these channels, MscK is unique in that its activation also requires external potassium ions. To better understand this dual gating mechanism by force and ligand, we elucidate distinct structures of MscK along the gating cycle using cryo-electron microscopy. The heptameric channel comprises three layers: a cytoplasmic domain, a periplasmic gating ring, and a markedly curved transmembrane domain that flattens and expands upon channel opening, which is accompanied by dilation of the periplasmic ring. Furthermore, our results support a potentially unifying mechanotransduction mechanism in ion channels depicted as flattening and expansion of the transmembrane domain., (© 2022. The Author(s).)
- Published
- 2022
- Full Text
- View/download PDF
21. Donor Macrophages Modulate Rejection After Heart Transplantation.
- Author
-
Kopecky BJ, Dun H, Amrute JM, Lin CY, Bredemeyer AL, Terada Y, Bayguinov PO, Koenig AL, Frye CC, Fitzpatrick JAJ, Kreisel D, and Lavine KJ
- Subjects
- Animals, Graft Rejection prevention & control, Humans, Macrophages, Mice, Mice, Inbred C57BL, Myeloid Differentiation Factor 88 genetics, Tissue Donors, Heart Transplantation adverse effects
- Abstract
Background: Cellular rejection after heart transplantation imparts significant morbidity and mortality. Current immunosuppressive strategies are imperfect, target recipient T cells, and have adverse effects. The innate immune response plays an essential role in the recruitment and activation of T cells. Targeting the donor innate immune response would represent the earliest interventional opportunity within the immune response cascade. There is limited knowledge about donor immune cell types and functions in the setting of cardiac transplantation, and no current therapeutics exist for targeting these cell populations., Methods: Using genetic lineage tracing, cell ablation, and conditional gene deletion, we examined donor mononuclear phagocyte diversity and macrophage function during acute cellular rejection of transplanted hearts in mice. We performed single-cell RNA sequencing on donor and recipient macrophages and monocytes at multiple time points after transplantation. On the basis of our imaging and single-cell RNA sequencing data, we evaluated the functional relevance of donor CCR2
+ (C-C chemokine receptor 2) and CCR2- macrophages using selective cell ablation strategies in donor grafts before transplant. Last, we performed functional validation that donor macrophages signal through MYD88 (myeloid differentiation primary response protein 88) to facilitate cellular rejection., Results: Donor macrophages persisted in the rejecting transplanted heart and coexisted with recipient monocyte-derived macrophages. Single-cell RNA sequencing identified donor CCR2+ and CCR2- macrophage populations and revealed remarkable diversity among recipient monocytes, macrophages, and dendritic cells. Temporal analysis demonstrated that donor CCR2+ and CCR2- macrophages were transcriptionally distinct, underwent significant morphologic changes, and displayed unique activation signatures after transplantation. Although selective depletion of donor CCR2- macrophages reduced allograft survival, depletion of donor CCR2+ macrophages prolonged allograft survival. Pathway analysis revealed that donor CCR2+ macrophages are activated through MYD88/nuclear factor kappa light chain enhancer of activated B cells signaling. Deletion of MYD88 in donor macrophages resulted in reduced antigen-presenting cell recruitment, reduced ability of antigen-presenting cells to present antigen to T cells, decreased emergence of allograft-reactive T cells, and extended allograft survival., Conclusions: Distinct populations of donor and recipient macrophages coexist within the transplanted heart. Donor CCR2+ macrophages are key mediators of allograft rejection, and deletion of MYD88 signaling in donor macrophages is sufficient to suppress rejection and extend allograft survival. This highlights the therapeutic potential of donor heart-based interventions.- Published
- 2022
- Full Text
- View/download PDF
22. Disease-associated mutations within the yeast DNAJB6 homolog Sis1 slow conformer-specific substrate processing and can be corrected by the modulation of nucleotide exchange factors.
- Author
-
Bhadra AK, Rau MJ, Daw JA, Fitzpatrick JAJ, Weihl CC, and True HL
- Subjects
- HSP40 Heat-Shock Proteins genetics, HSP40 Heat-Shock Proteins metabolism, HSP70 Heat-Shock Proteins genetics, HSP70 Heat-Shock Proteins metabolism, Humans, Molecular Chaperones genetics, Molecular Chaperones metabolism, Mutation, Nerve Tissue Proteins metabolism, Nucleotides metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Muscular Dystrophies, Limb-Girdle genetics, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Molecular chaperones, or heat shock proteins (HSPs), protect against the toxic misfolding and aggregation of proteins. As such, mutations or deficiencies within the chaperone network can lead to disease. Dominant mutations within DNAJB6 (Hsp40)-an Hsp70 co-chaperone-lead to a protein aggregation-linked myopathy termed Limb-Girdle Muscular Dystrophy Type D1 (LGMDD1). Here, we used the yeast prion model client in conjunction with in vitro chaperone activity assays to gain mechanistic insights into the molecular basis of LGMDD1. Here, we show how mutations analogous to those found in LGMDD1 affect Sis1 (a functional homolog of human DNAJB6) function by altering the structure of client protein aggregates, interfering with the Hsp70 ATPase cycle, dimerization and substrate processing; poisoning the function of wild-type protein. These results uncover the mechanisms through which LGMDD1-associated mutations alter chaperone activity, and provide insights relevant to potential therapeutic interventions., (© 2022. The Author(s).)
- Published
- 2022
- Full Text
- View/download PDF
23. Miswiring of Merkel cell and pruriceptive C fiber drives the itch-scratch cycle.
- Author
-
Feng J, Zhao Y, Xie Z, Zang K, Sviben S, Hu X, Fitzpatrick JAJ, Wen L, Liu Y, Wang T, Lawson K, Liu Q, Yan Y, Dong X, Han L, Wu GF, Kim BS, and Hu H
- Subjects
- Humans, Pruritus, Sensory Receptor Cells metabolism, Skin metabolism, Merkel Cells metabolism, Nerve Fibers, Unmyelinated metabolism
- Abstract
Itch sensation provokes the scratch reflex to protect us from harmful stimuli in the skin. Although scratching transiently relieves acute itch through activation of mechanoreceptors, it propagates the vicious itch-scratch cycle in chronic itch by further aggravating itch over time. Although well recognized clinically, the peripheral mechanisms underlying the itch-scratch cycle remain poorly understood. Here, we show that mechanical stimulation of the skin results in activation of the Piezo2 channels on Merkel cells that pathologically promotes spontaneous itch in experimental dry skin. Three-dimensional reconstruction and immunoelectron microscopy revealed structural alteration of MRGPRA3
+ pruriceptor nerve endings directed toward Merkel cells in the setting of dry skin. Our results uncover a functional miswiring mechanism under pathologic conditions, resulting in touch receptors triggering the firing of pruriceptors in the skin to drive the itch-scratch cycle.- Published
- 2022
- Full Text
- View/download PDF
24. Cryo-EM structure of the prothrombin-prothrombinase complex.
- Author
-
Ruben EA, Summers B, Rau MJ, Fitzpatrick JAJ, and Di Cera E
- Subjects
- Cryoelectron Microscopy, Factor V, Factor Va metabolism, Thromboplastin metabolism, Factor Xa metabolism, Prothrombin metabolism
- Abstract
The intrinsic and extrinsic pathways of the coagulation cascade converge to a common step where the prothrombinase complex, comprising the enzyme factor Xa (fXa), the cofactor fVa, Ca2+ and phospholipids, activates the zymogen prothrombin to the protease thrombin. The reaction entails cleavage at 2 sites, R271 and R320, generating the intermediates prethrombin 2 and meizothrombin, respectively. The molecular basis of these interactions that are central to hemostasis remains elusive. We solved 2 cryogenic electron microscopy (cryo-EM) structures of the fVa-fXa complex, 1 free on nanodiscs at 5.3-Å resolution and the other bound to prothrombin at near atomic 4.1-Å resolution. In the prothrombin-fVa-fXa complex, the Gla domains of fXa and prothrombin align on a plane with the C1 and C2 domains of fVa for interaction with membranes. Prothrombin and fXa emerge from this plane in curved conformations that bring their protease domains in contact with each other against the A2 domain of fVa. The 672ESTVMATRKMHDRLEPEDEE691 segment of the A2 domain closes on the protease domain of fXa like a lid to fix orientation of the active site. The 696YDYQNRL702 segment binds to prothrombin and establishes the pathway of activation by sequestering R271 against D697 and directing R320 toward the active site of fXa. The cryo-EM structure provides a molecular view of prothrombin activation along the meizothrombin pathway and suggests a mechanism for cleavage at the alternative R271 site. The findings advance our basic knowledge of a key step of coagulation and bear broad relevance to other interactions in the blood., (© 2022 by The American Society of Hematology.)
- Published
- 2022
- Full Text
- View/download PDF
25. Correction to: Size-Dependent Effective Diffusivity in Healthy Human and Porcine Joint Synovium.
- Author
-
Guang Y, Davis AL, McGrath TM, Pham CTN, Fitzpatrick JAJ, and Setton LA
- Published
- 2022
- Full Text
- View/download PDF
26. Epithelial Abnormalities in the Small Intestine of Zambian Children With Stunting.
- Author
-
Mulenga C, Sviben S, Chandwe K, Amadi B, Kayamba V, Fitzpatrick JAJ, Mudenda V, and Kelly P
- Abstract
Background: Environmental enteropathy (EE) contributes to impaired linear growth (stunting), in millions of children worldwide. We have previously reported that confocal laser endomicroscopy (CLE) shows fluorescein leaking from blood to gut lumen in vivo in adults and children with EE. We set out to identify epithelial lesions which might explain this phenomenon in Zambian children with stunting non-responsive to nutritional support., Methods: We performed confocal laser endomicroscopy (CLE) in 75 children and collected intestinal biopsies for histology in 91 children. CLE videos were evaluated, employing the Watson score to determine severity of leakiness. Morphometry was carried out on well-orientated mucosa and 3 biopsies were examined by electron microscopy., Results: Confocal laser endomicroscopy demonstrated substantial leakage from circulation to gut lumen in 73 (97%) children. Histology consistently showed characteristic changes of EE: villus blunting, lamina propria and epithelial inflammation, and depletion of secretory cells (Paneth cells and goblet cells). Epithelial abnormalities included marked variability in epithelial height, disorganised and shortened microvilli, dilated intercellular spaces, pseudostratification, formation of synechiae between epithelium on adjacent villi, crypt destruction, and abundant destructive lesions which may correspond to the microerosions identified on CLE., Conclusion: Epithelial abnormalities were almost universal in Zambian children with non-responsive stunting, including epithelial microerosions, cell-cell adhesion anomalies, and defects in secretory cells which may all contribute to impairment of mucosal barrier function and microbial translocation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mulenga, Sviben, Chandwe, Amadi, Kayamba, Fitzpatrick, Mudenda and Kelly.)
- Published
- 2022
- Full Text
- View/download PDF
27. Singlet Oxygen Leads to Structural Changes to Chloroplasts during their Degradation in the Arabidopsis thaliana plastid ferrochelatase two Mutant.
- Author
-
Fisher KE, Krishnamoorthy P, Joens MS, Chory J, Fitzpatrick JAJ, and Woodson JD
- Subjects
- Chloroplasts metabolism, Ferrochelatase, Gene Expression Regulation, Plant, Mutation genetics, Plastids metabolism, Singlet Oxygen metabolism, Arabidopsis genetics, Arabidopsis metabolism, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism
- Abstract
During stress, chloroplasts produce large amounts of reactive oxygen species (ROS). Chloroplasts also contain many nutrients, including 80% of a leaf's nitrogen supply. Therefore, to protect cells from photo-oxidative damage and to redistribute nutrients to sink tissues, chloroplasts are prime targets for degradation. Multiple chloroplast degradation pathways are induced by photo-oxidative stress or nutrient starvation, but the mechanisms by which damaged or senescing chloroplasts are identified, transported to the central vacuole and degraded are poorly defined. Here, we investigated the structures involved with degrading chloroplasts induced by the ROS singlet oxygen (1O2) in the Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant. Under mild 1O2 stress, most fc2 chloroplasts appeared normal, but had reduced starch content. A subset of chloroplasts was degrading, and some protruded into the central vacuole via 'blebbing' structures. A 3D electron microscopy analysis demonstrated that up to 35% of degrading chloroplasts contained such structures. While the location of a chloroplast within a cell did not affect the likelihood of its degradation, chloroplasts in spongy mesophyll cells were degraded at a higher rate than those in palisade mesophyll cells. To determine if degrading chloroplasts have unique structural characteristics, allowing them to be distinguished from healthy chloroplasts, we analyzed fc2 seedlings grown under different levels of photo-oxidative stress. A correlation was observed among chloroplast swelling, 1O2 signaling and the state of degradation. Finally, plastoglobule (PG) enzymes involved in chloroplast disassembly were upregulated while PGs increased their association with the thylakoid grana, implicating an interaction between 1O2-induced chloroplast degradation and senescence pathways., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2022
- Full Text
- View/download PDF
28. Hydraulic permeability and compressive properties of porcine and human synovium.
- Author
-
Rohanifar M, Johnston BB, Davis AL, Guang Y, Nommensen K, Fitzpatrick JAJ, Pham CN, and Setton LA
- Subjects
- Animals, Compressive Strength physiology, Elasticity, Humans, Models, Biological, Permeability, Stress, Mechanical, Swine, Synovial Membrane, Cartilage, Articular physiology
- Abstract
The synovium is a multilayer connective tissue separating the intra-articular spaces of the diarthrodial joint from the extra-synovial vascular and lymphatic supply. Synovium regulates drug transport into and out of the joint, yet its material properties remain poorly characterized. Here, we measured the compressive properties (aggregate modulus, Young's modulus, and Poisson's ratio) and hydraulic permeability of synovium with a combined experimental-computational approach. A compressive aggregate modulus and Young's modulus for the solid phase of synovium were quantified from linear regression of the equilibrium confined and unconfined compressive stress upon strain, respectively (H
A = 4.3 ± 2.0 kPa, Es = 2.1 ± 0.75, porcine; HA = 3.1 ± 2.0 kPa, Es = 2.8 ± 1.7, human). Poisson's ratio was estimated to be 0.39 and 0.40 for porcine and human tissue, respectively, from moduli values in a Monte Carlo simulation. To calculate hydraulic permeability, a biphasic finite element model's predictions were numerically matched to experimental data for the time-varying ramp and hold phase of a single increment of applied strain (k = 7.4 ± 4.1 × 10-15 m4 /N.s, porcine; k = 7.4 ± 4.3 × 10-15 m4 /N.s, human). We can use these newly measured properties to predict fluid flow gradients across the tissue in response to previously reported intra-articular pressures. These values for material constants are to our knowledge the first available measurements in synovium that are necessary to better understand drug transport in both healthy and pathological joints., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)- Published
- 2022
- Full Text
- View/download PDF
29. Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins.
- Author
-
Luke CJ, Markovina S, Good M, Wight IE, Thomas BJ, Linneman JM, Lanik WE, Koroleva O, Coffman MR, Miedel MT, Gong Q, Andress A, Campos Guerrero M, Wang S, Chen L, Beatty WL, Hausmann KN, White FV, Fitzpatrick JAJ, Orvedahl A, Pak SC, and Silverman GA
- Subjects
- Animals, Antigens, Neoplasm metabolism, Cell Line, Tumor, Humans, Mice, Serpins metabolism, Antigens, Neoplasm genetics, Cell Death, Epithelial Cells physiology, Serpins genetics
- Abstract
Lysosomal membrane permeabilization (LMP) and cathepsin release typifies lysosome-dependent cell death (LDCD). However, LMP occurs in most regulated cell death programs suggesting LDCD is not an independent cell death pathway, but is conscripted to facilitate the final cellular demise by other cell death routines. Previously, we demonstrated that Caenorhabditis elegans (C. elegans) null for a cysteine protease inhibitor, srp-6, undergo a specific LDCD pathway characterized by LMP and cathepsin-dependent cytoplasmic proteolysis. We designated this cell death routine, lysoptosis, to distinguish it from other pathways employing LMP. In this study, mouse and human epithelial cells lacking srp-6 homologues, mSerpinb3a and SERPINB3, respectively, demonstrated a lysoptosis phenotype distinct from other cell death pathways. Like in C. elegans, this pathway depended on LMP and released cathepsins, predominantly cathepsin L. These studies suggested that lysoptosis is an evolutionarily-conserved eukaryotic LDCD that predominates in the absence of neutralizing endogenous inhibitors., (© 2022. The Author(s).)
- Published
- 2022
- Full Text
- View/download PDF
30. Cryo-EM of CcsBA reveals the basis for cytochrome c biogenesis and heme transport.
- Author
-
Mendez DL, Lowder EP, Tillman DE, Sutherland MC, Collier AL, Rau MJ, Fitzpatrick JAJ, and Kranz RG
- Subjects
- Protein Transport, Cryoelectron Microscopy methods, Cytochromes c biosynthesis, Heme metabolism
- Abstract
Although the individual structures and respiratory functions of cytochromes are well studied, the structural basis for their assembly, including transport of heme for attachment, are unknown. We describe cryo-electron microscopy (cryo-EM) structures of CcsBA, a bifunctional heme transporter and cytochrome c (cyt c) synthase. Models built from the cryo-EM densities show that CcsBA is trapped with heme in two conformations, herein termed the closed and open states. The closed state has heme located solely at a transmembrane (TM) site, with a large periplasmic domain oriented such that access of heme to the cytochrome acceptor is denied. The open conformation contains two heme moieties, one in the TM-heme site and another in an external site (P-heme site). The presence of heme in the periplasmic site at the base of a chamber induces a large conformational shift that exposes the heme for reaction with apocytochrome c (apocyt c). Consistent with these structures, in vivo and in vitro cyt c synthase studies suggest a mechanism for transfer of the periplasmic heme to cytochrome., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)
- Published
- 2022
- Full Text
- View/download PDF
31. Structural mechanism of SARS-CoV-2 neutralization by two murine antibodies targeting the RBD.
- Author
-
Errico JM, Zhao H, Chen RE, Liu Z, Case JB, Ma M, Schmitz AJ, Rau MJ, Fitzpatrick JAJ, Shi PY, Diamond MS, Whelan SPJ, Ellebedy AH, and Fremont DH
- Subjects
- Angiotensin-Converting Enzyme 2 metabolism, Animals, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal immunology, Antibodies, Neutralizing chemistry, Antibodies, Viral chemistry, Cryoelectron Microscopy, Epitopes, B-Lymphocyte chemistry, Epitopes, B-Lymphocyte immunology, Humans, Mice, Protein Interaction Domains and Motifs immunology, Protein Structure, Quaternary, Spike Glycoprotein, Coronavirus chemistry, Antibodies, Neutralizing immunology, Antibodies, Viral immunology, COVID-19 immunology, SARS-CoV-2 immunology, Spike Glycoprotein, Coronavirus immunology
- Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has necessitated the rapid development of antibody-based therapies and vaccines as countermeasures. Here, we use cryoelectron microscopy (cryo-EM) to characterize two protective anti-SARS-CoV-2 murine monoclonal antibodies (mAbs) in complex with the spike protein, revealing similarities between epitopes targeted by human and murine B cells. The more neutralizing mAb, 2B04, binds the receptor-binding motif (RBM) of the receptor-binding domain (RBD) and competes with angiotensin-converting enzyme 2 (ACE2). By contrast, 2H04 binds adjacent to the RBM and does not compete for ACE2 binding. Naturally occurring sequence variants of SARS-CoV-2 and corresponding neutralization escape variants selected in vitro map to our structurally defined epitopes, suggesting that SARS-CoV-2 might evade therapeutic antibodies with a limited set of mutations, underscoring the importance of combination mAb therapeutics. Finally, we show that 2B04 neutralizes SARS-CoV-2 infection by preventing ACE2 engagement, whereas 2H04 reduces host cell attachment without directly disrupting ACE2-RBM interactions, providing distinct inhibitory mechanisms used by RBD-specific mAbs., Competing Interests: Declaration of interests D.H.F. is a founder of Courier Therapeutics. A.H.E. is a consultant for Inbios and Fimbrion Therapeutics. M.S.D. is a consultant for Inbios, Vir Biotechnology, and NGM Biopharmaceuticals and is on the Scientific Advisory Board of Moderna and Immunome. D.H.F., M.S.D., and A.H.E. have received unrelated funding support from Emergent BioSolutions. M.S.D. has sponsored research agreements from Moderna and Vir Biotechnology, A.H.E. has a sponsored research agreement from Abbvie, and D.H.F. has a sponsored research agreement from Mallinckrodt Pharmaceuticals., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
32. CHARGE syndrome protein CHD7 regulates epigenomic activation of enhancers in granule cell precursors and gyrification of the cerebellum.
- Author
-
Reddy NC, Majidi SP, Kong L, Nemera M, Ferguson CJ, Moore M, Goncalves TM, Liu HK, Fitzpatrick JAJ, Zhao G, Yamada T, Bonni A, and Gabel HW
- Subjects
- Animals, CHARGE Syndrome pathology, Cell Division genetics, Cerebellum pathology, Chromatin Assembly and Disassembly, DNA Helicases genetics, DNA-Binding Proteins genetics, Disease Models, Animal, Enhancer Elements, Genetic, Epigenesis, Genetic, Histone Code, Humans, Infant, Mice, Mice, Knockout, Mutation, Neural Stem Cells metabolism, Neurons metabolism, Polymicrogyria pathology, RNA-Seq, CHARGE Syndrome genetics, Cerebellum growth & development, DNA-Binding Proteins metabolism, Gene Expression Regulation, Developmental, Polymicrogyria genetics
- Abstract
Regulation of chromatin plays fundamental roles in the development of the brain. Haploinsufficiency of the chromatin remodeling enzyme CHD7 causes CHARGE syndrome, a genetic disorder that affects the development of the cerebellum. However, how CHD7 controls chromatin states in the cerebellum remains incompletely understood. Using conditional knockout of CHD7 in granule cell precursors in the mouse cerebellum, we find that CHD7 robustly promotes chromatin accessibility, active histone modifications, and RNA polymerase recruitment at enhancers. In vivo profiling of genome architecture reveals that CHD7 concordantly regulates epigenomic modifications associated with enhancer activation and gene expression of topologically-interacting genes. Genome and gene ontology studies show that CHD7-regulated enhancers are associated with genes that control brain tissue morphogenesis. Accordingly, conditional knockout of CHD7 triggers a striking phenotype of cerebellar polymicrogyria, which we have also found in a case of CHARGE syndrome. Finally, we uncover a CHD7-dependent switch in the preferred orientation of granule cell precursor division in the developing cerebellum, providing a potential cellular basis for the cerebellar polymicrogyria phenotype upon loss of CHD7. Collectively, our findings define epigenomic regulation by CHD7 in granule cell precursors and identify abnormal cerebellar patterning upon CHD7 depletion, with potential implications for our understanding of CHARGE syndrome., (© 2021. The Author(s).)
- Published
- 2021
- Full Text
- View/download PDF
33. Resident cardiac macrophages mediate adaptive myocardial remodeling.
- Author
-
Wong NR, Mohan J, Kopecky BJ, Guo S, Du L, Leid J, Feng G, Lokshina I, Dmytrenko O, Luehmann H, Bajpai G, Ewald L, Bell L, Patel N, Bredemeyer A, Weinheimer CJ, Nigro JM, Kovacs A, Morimoto S, Bayguinov PO, Fisher MR, Stump WT, Greenberg M, Fitzpatrick JAJ, Epelman S, Kreisel D, Sah R, Liu Y, Hu H, and Lavine KJ
- Subjects
- Animals, Cardiomyopathy, Dilated genetics, Cardiomyopathy, Dilated pathology, Humans, Mice, Mice, Inbred C57BL, Mice, Mutant Strains, Mutation, Myocardium metabolism, Troponin T genetics, Cardiomyopathy, Dilated metabolism, Macrophage Activation physiology, Macrophages metabolism, Ventricular Remodeling physiology
- Abstract
Cardiac macrophages represent a heterogeneous cell population with distinct origins, dynamics, and functions. Recent studies have revealed that C-C Chemokine Receptor 2 positive (CCR2
+ ) macrophages derived from infiltrating monocytes regulate myocardial inflammation and heart failure pathogenesis. Comparatively little is known about the functions of tissue resident (CCR2- ) macrophages. Herein, we identified an essential role for CCR2- macrophages in the chronically failing heart. Depletion of CCR2- macrophages in mice with dilated cardiomyopathy accelerated mortality and impaired ventricular remodeling and coronary angiogenesis, adaptive changes necessary to maintain cardiac output in the setting of reduced cardiac contractility. Mechanistically, CCR2- macrophages interacted with neighboring cardiomyocytes via focal adhesion complexes and were activated in response to mechanical stretch through a transient receptor potential vanilloid 4 (TRPV4)-dependent pathway that controlled growth factor expression. These findings establish a role for tissue-resident macrophages in adaptive cardiac remodeling and implicate mechanical sensing in cardiac macrophage activation., Competing Interests: Declaration of interests The authors have no financial or competing interests to disclose., (Copyright © 2021 Elsevier Inc. All rights reserved.)- Published
- 2021
- Full Text
- View/download PDF
34. Liver-Specific Deletion of Mouse Tm6sf2 Promotes Steatosis, Fibrosis, and Hepatocellular Cancer.
- Author
-
Newberry EP, Hall Z, Xie Y, Molitor EA, Bayguinov PO, Strout GW, Fitzpatrick JAJ, Brunt EM, Griffin JL, and Davidson NO
- Subjects
- Animals, Carcinoma, Hepatocellular metabolism, Carcinoma, Hepatocellular pathology, Fatty Liver metabolism, Lipidomics, Liver pathology, Liver Cirrhosis metabolism, Liver Neoplasms metabolism, Liver Neoplasms pathology, Mice, Mice, Knockout, Non-alcoholic Fatty Liver Disease genetics, Triglycerides metabolism, Carcinoma, Hepatocellular genetics, Fatty Liver genetics, Lipoproteins, VLDL metabolism, Liver metabolism, Liver Cirrhosis genetics, Liver Neoplasms genetics, Membrane Proteins genetics
- Abstract
Background and Aims: Human transmembrane 6 superfamily 2 (TM6SF2) variant rs58542926 is associated with NAFLD and HCC. However, conflicting reports in germline Tm6sf2 knockout mice suggest no change or decreased very low density lipoprotein (VLDL) secretion and either unchanged or increased hepatic steatosis, with no increased fibrosis. We generated liver-specific Tm6Sf2 knockout mice (Tm6 LKO) to study VLDL secretion and the impact on development and progression of NAFLD., Approach and Results: Two independent lines of Tm6 LKO mice exhibited spontaneous hepatic steatosis. Targeted lipidomic analyses showed increased triglyceride species whose distribution and abundance phenocopied findings in mice with liver-specific deletion of microsomal triglyceride transfer protein. The VLDL triglyceride secretion was reduced with small, underlipidated particles and unchanged or increased apolipoprotein B. Liver-specific adeno-associated viral, serotype 8 (AAV8) rescue using either wild-type or mutant E167K-Tm6 reduced hepatic steatosis and improved VLDL secretion. The Tm6 LKO mice fed a high milk-fat diet for 3 weeks exhibited increased steatosis and fibrosis, and those phenotypes were further exacerbated when mice were fed fibrogenic, high fat/fructose diets for 20 weeks. In two models of HCC, either neonatal mice injected with streptozotocin (NASH/STAM) and high-fat fed or with diethylnitrosamine injection plus fibrogenic diet feeding, Tm6 LKO mice exhibited increased steatosis, greater tumor burden, and increased tumor area versus Tm6 flox controls. Additionally, diethylnitrosamine-injected and fibrogenic diet-fed Tm6 LKO mice administered wild-type Tm6 or E167K-mutant Tm6 AAV8 revealed significant tumor attenuation, with tumor burden inversely correlated with Tm6 protein levels., Conclusions: Liver-specific Tm6sf2 deletion impairs VLDL secretion, promoting hepatic steatosis, fibrosis, and accelerated development of HCC, which was mitigated with AAV8- mediated rescue., (© 2021 by the American Association for the Study of Liver Diseases.)
- Published
- 2021
- Full Text
- View/download PDF
35. Heterogeneity of meningeal B cells reveals a lymphopoietic niche at the CNS borders.
- Author
-
Brioschi S, Wang WL, Peng V, Wang M, Shchukina I, Greenberg ZJ, Bando JK, Jaeger N, Czepielewski RS, Swain A, Mogilenko DA, Beatty WL, Bayguinov P, Fitzpatrick JAJ, Schuettpelz LG, Fronick CC, Smirnov I, Kipnis J, Shapiro VS, Wu GF, Gilfillan S, Cella M, Artyomov MN, Kleinstein SH, and Colonna M
- Subjects
- Aging, Animals, B-Lymphocyte Subsets immunology, Cell Movement, Central Nervous System physiology, Dura Mater immunology, Fibroblasts physiology, Homeostasis, Immune Privilege, Mice, Plasma Cells physiology, Single-Cell Analysis, B-Lymphocyte Subsets physiology, B-Lymphocytes physiology, Bone Marrow Cells physiology, Central Nervous System immunology, Dura Mater cytology, Lymphopoiesis, Meninges cytology, Meninges immunology, Skull anatomy & histology
- Abstract
The meninges contain adaptive immune cells that provide immunosurveillance of the central nervous system (CNS). These cells are thought to derive from the systemic circulation. Through single-cell analyses, confocal imaging, bone marrow chimeras, and parabiosis experiments, we show that meningeal B cells derive locally from the calvaria, which harbors a bone marrow niche for hematopoiesis. B cells reach the meninges from the calvaria through specialized vascular connections. This calvarial-meningeal path of B cell development may provide the CNS with a constant supply of B cells educated by CNS antigens. Conversely, we show that a subset of antigen-experienced B cells that populate the meninges in aging mice are blood-borne. These results identify a private source for meningeal B cells, which may help maintain immune privilege within the CNS., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)
- Published
- 2021
- Full Text
- View/download PDF
36. Maturation of Heterogeneity in Afferent Synapse Ultrastructure in the Mouse Cochlea.
- Author
-
Payne SA, Joens MS, Chung H, Skigen N, Frank A, Gattani S, Vaughn K, Schwed A, Nester M, Bhattacharyya A, Iyer G, Davis B, Carlquist J, Patel H, Fitzpatrick JAJ, and Rutherford MA
- Abstract
Auditory nerve fibers (ANFs) innervating the same inner hair cell (IHC) may have identical frequency tuning but different sound response properties. In cat and guinea pig, ANF response properties correlate with afferent synapse morphology and position on the IHC, suggesting a causal structure-function relationship. In mice, this relationship has not been fully characterized. Here we measured the emergence of synaptic morphological heterogeneities during maturation of the C57BL/6J mouse cochlea by comparing postnatal day 17 (p17, ∼3 days after hearing onset) with p34, when the mouse cochlea is mature. Using serial block face scanning electron microscopy and three-dimensional reconstruction we measured the size, shape, vesicle content, and position of 70 ribbon synapses from the mid-cochlea. Several features matured over late postnatal development. From p17 to p34, presynaptic densities (PDs) and post-synaptic densities (PSDs) became smaller on average (PDs: 0.75 to 0.33; PSDs: 0.58 to 0.31 μm
2 ) and less round as their short axes shortened predominantly on the modiolar side, from 770 to 360 nm. Membrane-associated synaptic vesicles decreased in number from 53 to 30 per synapse from p17 to p34. Anatomical coupling, measured as PSD to ribbon distance, tightened predominantly on the pillar side. Ribbons became less spherical as long-axes lengthened only on the modiolar side of the IHC, from 372 to 541 nm. A decreasing gradient of synaptic ribbon size along the modiolar-pillar axis was detected only at p34 after aligning synapses of adjacent IHCs to a common reference frame (median volumes in nm3 × 106 : modiolar 4.87; pillar 2.38). The number of ribbon-associated synaptic vesicles scaled with ribbon size (range 67 to 346 per synapse at p34), thus acquiring a modiolar-pillar gradient at p34, but overall medians were similar at p17 (120) and p34 (127), like ribbon surface area (0.36 vs. 0.34 μm2 ). PD and PSD morphologies were tightly correlated to each other at individual synapses, more so at p34 than p17, but not to ribbon morphology. These observations suggest that PDs and PSDs mature according to different cues than ribbons, and that ribbon size may be more influenced by cues from the IHC than the surrounding tissue., Competing Interests: MJ was employed by TESCAN. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Payne, Joens, Chung, Skigen, Frank, Gattani, Vaughn, Schwed, Nester, Bhattacharyya, Iyer, Davis, Carlquist, Patel, Fitzpatrick and Rutherford.)- Published
- 2021
- Full Text
- View/download PDF
37. Cryo-EM structures of human coagulation factors V and Va.
- Author
-
Ruben EA, Rau MJ, Fitzpatrick JAJ, and Di Cera E
- Subjects
- Humans, Protein Domains, Cryoelectron Microscopy, Factor Va chemistry, Factor Va ultrastructure
- Abstract
Coagulation factor V (fV) is the precursor of fVa, which, together with fXa, Ca2+, and phospholipids, defines the prothrombinase complex and activates prothrombin in the penultimate step of the coagulation cascade. We solved the cryogenic electron microscopy (cryo-EM) structures of human fV and fVa at atomic (3.3 Å) and near-atomic (4.4 Å) resolution, respectively. The structure of fV reveals the entire A1-A2-B-A3-C1-C2 assembly, but with a surprisingly disordered B domain. The C1 and C2 domains provide a platform for interaction with phospholipid membranes and support the A1 and A3 domains, with the A2 domain sitting on top of them. The B domain is highly dynamic and visible only for short segments connecting to the A2 and A3 domains. The A2 domain reveals all sites of proteolytic processing by thrombin and activated protein C, a partially buried epitope for binding fXa, and fully exposed epitopes for binding activated protein C and prothrombin. Removal of the B domain and activation to fVa exposes the sites of cleavage by activated protein C at R306 and R506 and produces increased disorder in the A1-A2-A3-C1-C2 assembly, especially in the C-terminal acidic portion of the A2 domain that is responsible for prothrombin binding. Ordering of this region and full exposure of the fXa epitope emerge as necessary steps in the assembly of the prothrombin-prothrombinase complex. These structures offer molecular context for the function of fV and fVa and pioneer the analysis of coagulation factors by cryo-EM., (© 2021 by The American Society of Hematology.)
- Published
- 2021
- Full Text
- View/download PDF
38. Targeted Therapy to β3 Integrin Reduces Chemoresistance in Breast Cancer Bone Metastases.
- Author
-
Fox GC, Su X, Davis JL, Xu Y, Kwakwa KA, Ross MH, Fontana F, Xiang J, Esser AK, Cordell E, Pagliai K, Dang HX, Sivapackiam J, Stewart SA, Maher CA, Bakewell SJ, Fitzpatrick JAJ, Sharma V, Achilefu S, Veis DJ, Lanza GM, and Weilbaecher KN
- Subjects
- Animals, Antineoplastic Agents pharmacology, Bone Neoplasms metabolism, Breast Neoplasms metabolism, Breast Neoplasms pathology, Cell Line, Tumor, Docetaxel pharmacology, Drug Resistance, Neoplasm, Female, Humans, Mice, Mice, Inbred C57BL, Molecular Targeted Therapy, Survival Analysis, Bone Neoplasms drug therapy, Bone Neoplasms secondary, Breast Neoplasms drug therapy, Integrin beta3 metabolism
- Abstract
Breast cancer bone metastases are common and incurable. Tumoral integrin β3 (β3) expression is induced through interaction with the bone microenvironment. Although β3 is known to promote bone colonization, its functional role during therapy of established bone metastases is not known. We found increased numbers of β3
+ tumor cells in murine bone metastases after docetaxel chemotherapy. β3+ tumor cells were present in 97% of post-neoadjuvant chemotherapy triple-negative breast cancer patient samples ( n = 38). High tumoral β3 expression was associated with worse outcomes in both pre- and postchemotherapy triple-negative breast cancer groups. Genetic deletion of tumoral β3 had minimal effect in vitro , but significantly enhanced in vivo docetaxel activity, particularly in the bone. Rescue experiments confirmed that this effect required intact β3 signaling. Ultrastructural, transcriptomic, and functional analyses revealed an alternative metabolic response to chemotherapy in β3-expressing cells characterized by enhanced oxygen consumption, reactive oxygen species generation, and protein production. We identified mTORC1 as a candidate for therapeutic targeting of this β3-mediated, chemotherapy-induced metabolic response. mTORC1 inhibition in combination with docetaxel synergistically attenuated murine bone metastases. Furthermore, micelle nanoparticle delivery of mTORC1 inhibitor to cells expressing activated αvβ3 integrins enhanced docetaxel efficacy in bone metastases. Taken together, we show that β3 integrin induction by the bone microenvironment promotes resistance to chemotherapy through an altered metabolic response that can be defused by combination with αvβ3-targeted mTORC1 inhibitor nanotherapy. Our work demonstrates the importance of the metastatic microenvironment when designing treatments and presents new, bone-specific strategies for enhancing chemotherapeutic efficacy., (©2021 American Association for Cancer Research.)- Published
- 2021
- Full Text
- View/download PDF
39. Size-Dependent Effective Diffusivity in Healthy Human and Porcine Joint Synovium.
- Author
-
Guang Y, Davis AL, McGrath TM, Pham CTN, Fitzpatrick JAJ, and Setton LA
- Subjects
- Animals, Diffusion, Humans, Knee Joint metabolism, Molecular Weight, Swine, Models, Biological, Pharmaceutical Preparations chemistry, Pharmaceutical Preparations metabolism, Synovial Membrane metabolism
- Abstract
Intra-articular drug delivery can be effective in targeting a diseased joint but is hampered by rapid clearance times from the diarthrodial joint. The synovium is a multi-layered tissue that surrounds the diarthrodial joint and governs molecular transport into and out of the joint. No models of drug clearance through synovium exist to quantify diffusivity across solutes, tissue type and disease pathology. We previously have developed a finite element model of synovium as a porous, permeable, fluid-filled tissue and used an inverse method to determine urea's effective diffusivity (D
eff ) in de-vitalized synovium explants.22 Here we apply this method to determine Deff from unsteady diffusive transport of model solutes and confirm the role of molecular weight in solute transport. As molecular weight increased, Deff decreased in both human and porcine tissues, with similar behavior across the two species. Unsteady transport was well-described by a single exponential transient decay in concentration, yielding solute half-lives (t1/2 ) that compared favorably with the Deff determined from the finite element model fit. Determined values for Deff parallel prior observations of size-dependent in vivo drug clearance and provide an intrinsic parameter with greater ability to resolve size-dependence in vitro. Thus, this work forms the basis for understanding the influence of size on drug transport in synovium and can guide future studies to elucidate the role of charge and tissue pathology on the transport of therapeutics in healthy and pathological human synovium.- Published
- 2021
- Full Text
- View/download PDF
40. Cryo-EM structure of the Rous sarcoma virus octameric cleaved synaptic complex intasome.
- Author
-
Pandey KK, Bera S, Shi K, Rau MJ, Oleru AV, Fitzpatrick JAJ, Engelman AN, Aihara H, and Grandgenett DP
- Subjects
- DNA, Viral metabolism, HIV Integrase ultrastructure, Integrase Inhibitors pharmacology, Integrases metabolism, Molecular Docking Simulation, Protein Conformation, Protein Multimerization, Rous sarcoma virus drug effects, Rous sarcoma virus enzymology, Rous sarcoma virus genetics, Virus Replication, Cryoelectron Microscopy, DNA, Viral ultrastructure, Integrases ultrastructure, Rous sarcoma virus ultrastructure, Single Molecule Imaging, Virus Integration drug effects
- Abstract
Despite conserved catalytic integration mechanisms, retroviral intasomes composed of integrase (IN) and viral DNA possess diverse structures with variable numbers of IN subunits. To investigate intasome assembly mechanisms, we employed the Rous sarcoma virus (RSV) IN dimer that assembles a precursor tetrameric structure in transit to the mature octameric intasome. We determined the structure of RSV octameric intasome stabilized by a HIV-1 IN strand transfer inhibitor using single particle cryo-electron microscopy. The structure revealed significant flexibility of the two non-catalytic distal IN dimers along with previously unrecognized movement of the conserved intasome core, suggesting ordered conformational transitions between intermediates that may be important to capture the target DNA. Single amino acid substitutions within the IN C-terminal domain affected intasome assembly and function in vitro and infectivity of pseudotyped RSV virions. Unexpectedly, 17 C-terminal amino acids of IN were dispensable for virus infection despite regulating the transition of the tetrameric intasome to the octameric form in vitro. We speculate that this region may regulate the binding of highly flexible distal IN dimers to the intasome core to form the octameric complex. Our studies reveal key steps in the assembly of RSV intasomes.
- Published
- 2021
- Full Text
- View/download PDF
41. Cryo-EM structure of a proton-activated chloride channel TMEM206.
- Author
-
Deng Z, Zhao Y, Feng J, Zhang J, Zhao H, Rau MJ, Fitzpatrick JAJ, Hu H, and Yuan P
- Abstract
TMEM206 has been recently identified as an evolutionarily conserved chloride channel that underlies ubiquitously expressed, proton-activated, outwardly rectifying anion currents. Here, we report the cryo-electron microscopy structure of pufferfish TMEM206, which forms a trimeric channel, with each subunit comprising two transmembrane segments and a large extracellular domain. An ample vestibule in the extracellular region is accessible laterally from the three side portals. The central pore contains multiple constrictions. A conserved lysine residue near the cytoplasmic end of the inner helix forms the presumed chloride ion selectivity filter. Unprecedentedly, the core structure and assembly closely resemble those of the epithelial sodium channel/degenerin family of sodium channels that are unrelated in amino acid sequence and conduct cations instead of anions. Together with electrophysiology, this work provides insights into ion conduction and gating for a new class of chloride channels that is architecturally distinct from previously characterized chloride channel families., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)
- Published
- 2021
- Full Text
- View/download PDF
42. Pancreatic β-Cell-Specific Deletion of VPS41 Causes Diabetes Due to Defects in Insulin Secretion.
- Author
-
Burns CH, Yau B, Rodriguez A, Triplett J, Maslar D, An YS, van der Welle REN, Kossina RG, Fisher MR, Strout GW, Bayguinov PO, Veenendaal T, Chitayat D, Fitzpatrick JAJ, Klumperman J, Kebede MA, and Asensio CS
- Subjects
- Animals, Cell Line, Diabetes Mellitus genetics, Exocytosis physiology, Glucose Tolerance Test, Mice, Mice, Knockout, Rats, Vesicular Transport Proteins genetics, Diabetes Mellitus metabolism, Insulin Secretion genetics, Insulin-Secreting Cells metabolism, Secretory Vesicles metabolism, Vesicular Transport Proteins metabolism
- Abstract
Insulin secretory granules (SGs) mediate the regulated secretion of insulin, which is essential for glucose homeostasis. The basic machinery responsible for this regulated exocytosis consists of specific proteins present both at the plasma membrane and on insulin SGs. The protein composition of insulin SGs thus dictates their release properties, yet the mechanisms controlling insulin SG formation, which determine this molecular composition, remain poorly understood. VPS41, a component of the endolysosomal tethering homotypic fusion and vacuole protein sorting (HOPS) complex, was recently identified as a cytosolic factor involved in the formation of neuroendocrine and neuronal granules. We now find that VPS41 is required for insulin SG biogenesis and regulated insulin secretion. Loss of VPS41 in pancreatic β-cells leads to a reduction in insulin SG number, changes in their transmembrane protein composition, and defects in granule-regulated exocytosis. Exploring a human point mutation, identified in patients with neurological but no endocrine defects, we show that the effect on SG formation is independent of HOPS complex formation. Finally, we report that mice with a deletion of VPS41 specifically in β-cells develop diabetes due to severe depletion of insulin SG content and a defect in insulin secretion. In sum, our data demonstrate that VPS41 contributes to glucose homeostasis and metabolism., (© 2020 by the American Diabetes Association.)
- Published
- 2021
- Full Text
- View/download PDF
43. Liver-specific deletion of Mttp versus Tm6sf2 reveals distinct defects in stepwise VLDL assembly.
- Author
-
Newberry EP, Strout GW, Fitzpatrick JAJ, and Davidson NO
- Subjects
- Animals, Mice, Gene Deletion, Liver metabolism, Membrane Proteins genetics, Membrane Proteins metabolism, Lipoproteins, VLDL metabolism
- Published
- 2021
- Full Text
- View/download PDF
44. Assaying three-dimensional cellular architecture using X-ray tomographic and correlated imaging approaches.
- Author
-
Bayguinov PO, Fisher MR, and Fitzpatrick JAJ
- Subjects
- Contrast Media chemistry, Microscopy, Electron, Scanning, Nanoparticles chemistry, Saccharomyces cerevisiae ultrastructure, Tomography, X-Ray instrumentation, X-Ray Microtomography, Imaging, Three-Dimensional methods, Tomography, X-Ray methods
- Abstract
Much of our understanding of the spatial organization of and interactions between cellular organelles and macromolecular complexes has been the result of imaging studies utilizing either light- or electron-based microscopic analyses. These classical approaches, while insightful, are nonetheless limited either by restrictions in resolution or by the sheer complexity of generating multidimensional data. Recent advances in the use and application of X-rays to acquire micro- and nanotomographic data sets offer an alternative methodology to visualize cellular architecture at the nanoscale. These new approaches allow for the subcellular analyses of unstained vitrified cells and three-dimensional localization of specific protein targets and have served as an essential tool in bridging light and electron correlative microscopy experiments. Here, we review the theory, instrumentation details, acquisition principles, and applications of both soft X-ray tomography and X-ray microscopy and how the use of these techniques offers a succinct means of analyzing three-dimensional cellular architecture. We discuss some of the recent work that has taken advantage of these approaches and detail how they have become integral in correlative microscopy workflows., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Bayguinov et al.)
- Published
- 2020
- Full Text
- View/download PDF
45. Structural mechanism for gating of a eukaryotic mechanosensitive channel of small conductance.
- Author
-
Deng Z, Maksaev G, Schlegel AM, Zhang J, Rau M, Fitzpatrick JAJ, Haswell ES, and Yuan P
- Subjects
- Arabidopsis Proteins ultrastructure, Escherichia coli Proteins chemistry, Escherichia coli Proteins metabolism, Ion Channels ultrastructure, Models, Molecular, Mutant Proteins chemistry, Mutant Proteins metabolism, Protein Domains, Protein Structure, Secondary, Arabidopsis metabolism, Arabidopsis Proteins chemistry, Arabidopsis Proteins metabolism, Eukaryota metabolism, Ion Channel Gating, Ion Channels chemistry, Ion Channels metabolism, Mechanotransduction, Cellular
- Abstract
Mechanosensitive ion channels transduce physical force into electrochemical signaling that underlies an array of fundamental physiological processes, including hearing, touch, proprioception, osmoregulation, and morphogenesis. The mechanosensitive channels of small conductance (MscS) constitute a remarkably diverse superfamily of channels critical for management of osmotic pressure. Here, we present cryo-electron microscopy structures of a MscS homolog from Arabidopsis thaliana, MSL1, presumably in both the closed and open states. The heptameric MSL1 channel contains an unusual bowl-shaped transmembrane region, which is reminiscent of the evolutionarily and architecturally unrelated mechanosensitive Piezo channels. Upon channel opening, the curved transmembrane domain of MSL1 flattens and expands. Our structures, in combination with functional analyses, delineate a structural mechanism by which mechanosensitive channels open under increased membrane tension. Further, the shared structural feature between unrelated channels suggests the possibility of a unified mechanical gating mechanism stemming from membrane deformation induced by a non-planar transmembrane domain.
- Published
- 2020
- Full Text
- View/download PDF
46. Gating of human TRPV3 in a lipid bilayer.
- Author
-
Deng Z, Maksaev G, Rau M, Xie Z, Hu H, Fitzpatrick JAJ, and Yuan P
- Subjects
- Cryoelectron Microscopy, Lipid Bilayers metabolism, Models, Molecular, Mutation, Nanostructures chemistry, Protein Conformation, TRPV Cation Channels genetics, Ion Channel Gating physiology, Lipid Bilayers chemistry, TRPV Cation Channels chemistry, TRPV Cation Channels metabolism
- Abstract
The transient receptor potential cation channel subfamily V member 3 (TRPV3) channel plays a critical role in skin physiology, and mutations in TRPV3 result in the development of a congenital skin disorder, Olmsted syndrome. Here we describe multiple cryo-electron microscopy structures of human TRPV3 reconstituted into lipid nanodiscs, representing distinct functional states during the gating cycle. The ligand-free, closed conformation reveals well-ordered lipids interacting with the channel and two physical constrictions along the ion-conduction pore involving both the extracellular selectivity filter and intracellular helix bundle crossing. Both the selectivity filter and bundle crossing expand upon activation, accompanied by substantial structural rearrangements at the cytoplasmic intersubunit interface. Transition to the inactivated state involves a secondary structure change of the pore-lining helix, which contains a π-helical segment in the closed and open conformations, but becomes entirely α-helical upon inactivation. Together with electrophysiological characterization, structures of TRPV3 in a lipid membrane environment provide unique insights into channel activation and inactivation mechanisms.
- Published
- 2020
- Full Text
- View/download PDF
47. Cryo-EM structures of the ATP release channel pannexin 1.
- Author
-
Deng Z, He Z, Maksaev G, Bitter RM, Rau M, Fitzpatrick JAJ, and Yuan P
- Subjects
- Adenosine Triphosphate genetics, Animals, Anura genetics, Cell Membrane chemistry, Cell Membrane genetics, Connexins chemistry, Connexins genetics, Humans, Nerve Tissue Proteins chemistry, Nerve Tissue Proteins genetics, Protein Conformation, Signal Transduction genetics, Adenosine Triphosphate chemistry, Cell Membrane ultrastructure, Connexins ultrastructure, Cryoelectron Microscopy, Nerve Tissue Proteins ultrastructure
- Abstract
The plasma membrane adenosine triphosphate (ATP) release channel pannexin 1 (PANX1) has been implicated in many physiological and pathophysiological processes associated with purinergic signaling, including cancer progression, apoptotic cell clearance, inflammation, blood pressure regulation, oocyte development, epilepsy and neuropathic pain. Here we present near-atomic-resolution structures of human and frog PANX1 determined by cryo-electron microscopy that revealed a heptameric channel architecture. Compatible with ATP permeation, the transmembrane pore and cytoplasmic vestibule were exceptionally wide. An extracellular tryptophan ring located at the outer pore created a constriction site, potentially functioning as a molecular sieve that restricts the size of permeable substrates. The amino and carboxyl termini, not resolved in the density map, appeared to be structurally dynamic and might contribute to narrowing of the pore during channel gating. In combination with functional characterization, this work elucidates the previously unknown architecture of pannexin channels and establishes a foundation for understanding their unique channel properties.
- Published
- 2020
- Full Text
- View/download PDF
48. Combined Experimental Approach and Finite Element Modeling of Small Molecule Transport Through Joint Synovium to Measure Effective Diffusivity.
- Author
-
Guang Y, McGrath TM, Klug NR, Nims RJ, Shih CC, Bayguinov PO, Guilak F, Pham CTN, Fitzpatrick JAJ, and Setton LA
- Abstract
Trans-synovial solute transport plays a critical role in the clearance of intra-articularly (IA) delivered drugs. In this study, we present a computational finite element model (FEM) of solute transport through the synovium validated by experiments on synovial explants. Unsteady diffusion of urea, a small uncharged molecule, was measured through devitalized porcine and human synovium using custom-built diffusion chambers. A multiphasic computational model was constructed and optimized with the experimental data to extract effective diffusivity for urea within the synovium. A monotonic decrease in urea concentration was observed in the donor bath over time, with an effective diffusivity found to be an order of magnitude lower in synovium versus that measured in free solution. Parametric studies incorporating an intimal cell layer with varying thickness and varying effective diffusivities were performed, revealing a dependence of drug clearance kinetics on both parameters. The findings of this study indicate that the synovial matrix impedes urea solute transport out of the joint with little retention of the solute in the matrix., (Copyright © 2020 by ASME.)
- Published
- 2020
- Full Text
- View/download PDF
49. Cryo-EM structure of a neuronal functional amyloid implicated in memory persistence in Drosophila .
- Author
-
Hervas R, Rau MJ, Park Y, Zhang W, Murzin AG, Fitzpatrick JAJ, Scheres SHW, and Si K
- Subjects
- Animals, Cryoelectron Microscopy, Drosophila melanogaster, Glutamine chemistry, Hydrophobic and Hydrophilic Interactions, Protein Conformation, Amyloid chemistry, Drosophila Proteins chemistry, Memory, Long-Term, Neurons metabolism, Protein Aggregates, RNA-Binding Proteins chemistry, Transcription Factors chemistry, mRNA Cleavage and Polyadenylation Factors chemistry
- Abstract
How long-lived memories withstand molecular turnover is a fundamental question. Aggregates of a prion-like RNA-binding protein, cytoplasmic polyadenylation element-binding (CPEB) protein, is a putative substrate of long-lasting memories. We isolated aggregated Drosophila CPEB, Orb2, from adult heads and determined its activity and atomic structure, at 2.6-angstrom resolution, using cryo-electron microscopy. Orb2 formed ~75-nanometer-long threefold-symmetric amyloid filaments. Filament formation transformed Orb2 from a translation repressor to an activator and "seed" for further translationally active aggregation. The 31-amino acid protofilament core adopted a cross-β unit with a single hydrophilic hairpin stabilized through interdigitated glutamine packing. Unlike the hydrophobic core of pathogenic amyloids, the hydrophilic core of Orb2 filaments suggests how some neuronal amyloids could be a stable yet regulatable substrate of memory., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)
- Published
- 2020
- Full Text
- View/download PDF
50. Staphylococcus aureus Infects Osteoclasts and Replicates Intracellularly.
- Author
-
Krauss JL, Roper PM, Ballard A, Shih CC, Fitzpatrick JAJ, Cassat JE, Ng PY, Pavlos NJ, and Veis DJ
- Subjects
- Animals, Bacterial Proteins genetics, Bacterial Proteins metabolism, Cell Differentiation, Cells, Cultured, Female, Macrophages metabolism, Male, Mice, Osteoblasts microbiology, Osteomyelitis metabolism, Osteomyelitis microbiology, Phagosomes metabolism, RANK Ligand metabolism, Staphylococcus aureus drug effects, Osteoclasts microbiology, Staphylococcus aureus metabolism, Staphylococcus aureus pathogenicity
- Abstract
Osteomyelitis (OM), or inflammation of bone tissue, occurs most frequently as a result of bacterial infection and severely perturbs bone structure. OM is predominantly caused by Staphylococcus aureus , and even with proper treatment, OM has a high rate of recurrence and chronicity. While S. aureus has been shown to infect osteoblasts, it remains unclear whether osteoclasts (OCs) are also a target of intracellular infection. Here, we demonstrate the ability of S. aureus to intracellularly infect and divide within OCs. OCs were differentiated from bone marrow macrophages (BMMs) by exposure to receptor activator of nuclear factor kappa-B ligand (RANKL). By utilizing an intracellular survival assay and flow cytometry, we found that at 18 h postinfection the intracellular burden of S. aureus increased dramatically in cells with at least 2 days of RANKL exposure, while the bacterial burden decreased in BMMs. To further explore the signals downstream of RANKL, we manipulated factors controlling OC differentiation, NFATc1 and alternative NF-κB, and found that intracellular bacterial growth correlates with NFATc1 levels in RANKL-treated cells. Confocal and time-lapse microscopy in mature OCs showed a range of intracellular infection that correlated inversely with S. aureus -phagolysosome colocalization. The propensity of OCs to become infected, paired with their diminished bactericidal capacity compared to BMMs, could promote OM progression by allowing S. aureus to evade initial immune regulation and proliferate at the periphery of lesions where OCs are most abundant. IMPORTANCE The inflammation of bone tissue is called osteomyelitis, and most cases are caused by an infection with the bacterium Staphylococcus aureus To date, the bone-building cells, osteoblasts, have been implicated in the progression of these infections, but not much is known about how the bone-resorbing cells, osteoclasts, participate. In this study, we show that S. aureus can infect osteoclasts and proliferate inside these cells, whereas bone-residing macrophages, immune cells related to osteoclasts, destroy the bacteria. These findings elucidate a unique role for osteoclasts to harbor bacteria during infection, providing a possible mechanism by which bacteria could evade destruction by the immune system., (Copyright © 2019 Krauss et al.)
- Published
- 2019
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.