scholarly journals ESCRT-III acts in scissioning new peroxisomes from the ER

2017 ◽  
Author(s):  
Fred D. Mast ◽  
Thurston Herricks ◽  
Kathleen M. Strehler ◽  
Leslie R. Miller ◽  
Ramsey A. Saleem ◽  
...  
Keyword(s):  
De Novo ◽  
Genetic Disorders ◽  
Dynamic Control ◽  
Eukaryotic Cell ◽  
Peroxisome Biogenesis ◽  
Endosomal Sorting ◽  
Obesity And Diabetes ◽  
A Cell ◽  
And Function

AbstractDynamic control of peroxisome proliferation is integral to the peroxisome’s many functions. A breakdown in the ability of cells to form peroxisomes is linked to many human health issues, including defense against infectious agents, cancer, aging, heart disease, obesity and diabetes, and forms the basis of a spectrum of peroxisomal genetic disorders that cause severe neuropathologies. The ER serves as a source for preperoxisomal vesicles (PPVs) that mature into peroxisomes during de novo peroxisome biogenesis and to support growth and division of existing peroxisomes. However, the mechanism of PPV formation and release from the ER remains poorly understood. Here we show that the evolutionarily ancient endosomal sorting complexes required for transport (ESCRT)-III are peroxisome biogenesis factors that function to cleave PPVs budding from the ER into the cytosol. Using comprehensive morphological and genetic assays of peroxisome formation and function we find that absence of ESCRT-III proteins impedes de novo peroxisome formation and results in an aberrant peroxisome population in vivo. Using a cell-free PPV budding assay we show that ESCRT-III proteins Vps20 and Snf7 are required to release PPVs from the ER. ESCRT-III is therefore a positive effector of membrane scission for vesicles budding both away from and towards the cytosol, a finding that has important implications for the evolutionary timing of emergence of peroxisomes and the rest of the internal membrane architecture of the eukaryotic cell.

scholarly journals ESCRT-III is required for scissioning new peroxisomes from the endoplasmic reticulum

2018 ◽  
Vol 217 (6) ◽  
pp. 2087-2102 ◽  
Author(s):  
Fred D. Mast ◽  
Thurston Herricks ◽  
Kathleen M. Strehler ◽  
Leslie R. Miller ◽  
Ramsey A. Saleem ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
De Novo ◽  
Dynamic Control ◽  
Eukaryotic Cell ◽  
Peroxisome Proliferation ◽  
Peroxisome Biogenesis ◽  
Endosomal Sorting ◽  
Internal Membrane ◽  
A Cell

Dynamic control of peroxisome proliferation is integral to the peroxisome’s many functions. The endoplasmic reticulum (ER) serves as a source of preperoxisomal vesicles (PPVs) that mature into peroxisomes during de novo peroxisome biogenesis and support growth and division of existing peroxisomes. However, the mechanism of PPV formation and release from the ER remains poorly understood. In this study, we show that endosomal sorting complexes required for transport (ESCRT)-III are required to release PPVs budding from the ER into the cytosol. Absence of ESCRT-III proteins impedes de novo peroxisome formation and results in an aberrant peroxisome population in vivo. Using a cell-free PPV budding assay, we show that ESCRT-III proteins Vps20 and Snf7 are necessary to release PPVs from the ER. ESCRT-III is therefore a positive effector of membrane scission for vesicles budding both away from and toward the cytosol. These findings have important implications for the evolutionary timing of emergence of peroxisomes and the rest of the internal membrane architecture of the eukaryotic cell.

scholarly journals Analysis of the roles of phosphatidylinositol-4,5-bisphosphate and individual subunits in assembly, localization, and function of Saccharomyces cerevisiae target of rapamycin complex 2

2019 ◽  
Vol 30 (12) ◽  
pp. 1555-1574 ◽  
Author(s):  
Maria Nieves Martinez Marshall ◽  
Anita Emmerstorfer-Augustin ◽  
Kristin L. Leskoske ◽  
Lydia H. Zhang ◽  
Biyun Li ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Lipid Metabolism ◽  
Eukaryotic Cell ◽  
Target Of Rapamycin ◽  
Multiprotein Complex ◽  
Ph Domain ◽  
Evolutionarily Conserved ◽  
And Function

Eukaryotic cell survival requires maintenance of plasma membrane (PM) homeostasis in response to environmental insults and changes in lipid metabolism. In yeast, a key regulator of PM homeostasis is target of rapamycin (TOR) complex 2 (TORC2), a multiprotein complex containing the evolutionarily conserved TOR protein kinase isoform Tor2. PM localization is essential for TORC2 function. One core TORC2 subunit (Avo1) and two TORC2-­associated regulators (Slm1 and Slm2) contain pleckstrin homology (PH) domains that exhibit specificity for binding phosphatidylinositol-4,5- bisphosphate (PtdIns4,5P2). To investigate the roles of PtdIns4,5P2 and constituent subunits of TORC2, we used auxin-inducible degradation to systematically eliminate these factors and then examined localization, association, and function of the remaining TORC2 components. We found that PtdIns4,5P2 depletion significantly reduced TORC2 activity, yet did not prevent PM localization or cause disassembly of TORC2. Moreover, truncated Avo1 (lacking its C-terminal PH domain) was still recruited to the PM and supported growth. Even when all three PH-containing proteins were absent, the remaining TORC2 subunits were PM-bound. Revealingly, Avo3 localized to the PM independent of both Avo1 and Tor2, whereas both Tor2 and Avo1 required Avo3 for their PM anchoring. Our findings provide new mechanistic information about TORC2 and pinpoint Avo3 as pivotal for TORC2 PM localization and assembly in vivo.

scholarly journals Functional partitioning of a liquid-like organelle during assembly of axonemal dyneins

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Chanjae Lee ◽  
Rachael M Cox ◽  
Ophelia Papoulas ◽  
Amjad Horani ◽  
Kevin Drew ◽  
...  
Keyword(s):  
Affinity Purification ◽  
Human Genetic Disease ◽  
Interaction Partners ◽  
A Cell ◽  
Cell Type Specific ◽  
Axonemal Dynein ◽  
And Function ◽  
Affinity Purification Mass Spectrometry ◽  
Functional Partitioning

Ciliary motility is driven by axonemal dyneins that are assembled in the cytoplasm before deployment to cilia. Motile ciliopathy can result from defects in the dyneins themselves or from defects in factors required for their cytoplasmic pre-assembly. Recent work demonstrates that axonemal dyneins, their specific assembly factors, and broadly-acting chaperones are concentrated in liquid-like organelles in the cytoplasm called DynAPs (Dynein Axonemal Particles). Here, we use in vivo imaging in Xenopus to show that inner dynein arm (IDA) and outer dynein arm (ODA) subunits are partitioned into non-overlapping sub-regions within DynAPs. Using affinity- purification mass-spectrometry of in vivo interaction partners, we also identify novel partners for inner and outer dynein arms. Among these, we identify C16orf71/Daap1 as a novel axonemal dynein regulator. Daap1 interacts with ODA subunits, localizes specifically to the cytoplasm, is enriched in DynAPs, and is required for the deployment of ODAs to axonemes. Our work reveals a new complexity in the structure and function of a cell-type specific liquid-like organelle that is directly relevant to human genetic disease.

scholarly journals Emergence and function of cortical offset responses in sound termination detection

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Magdalena Solyga ◽  
Tania Rinaldi Barkat
Keyword(s):  
Auditory Cortex ◽  
Auditory System ◽  
Auditory Processing ◽  
De Novo ◽  
Central Auditory System ◽  
In Vivo Electrophysiology ◽  
Termination Detection ◽  
Peripheral Auditory System ◽  
And Function

Offset responses in auditory processing appear after a sound terminates. They arise in neuronal circuits within the peripheral auditory system, but their role in the central auditory system remains unknown. Here, we ask what the behavioral relevance of cortical offset responses is and what circuit mechanisms drive them. At the perceptual level, our results reveal that experimentally minimizing auditory cortical offset responses decreases the mouse performance to detect sound termination, assigning a behavioral role to offset responses. By combining in vivo electrophysiology in the auditory cortex and thalamus of awake mice, we also demonstrate that cortical offset responses are not only inherited from the periphery but also amplified and generated de novo. Finally, we show that offset responses code more than silence, including relevant changes in sound trajectories. Together, our results reveal the importance of cortical offset responses in encoding sound termination and detecting changes within temporally discontinuous sounds crucial for speech and vocalization.

scholarly journals Structural basis of altered potency and efficacy displayed by a major in vivo metabolite of the anti-diabetic PPARγ drug pioglitazone

2018 ◽  
Author(s):  
Sarah A. Mosure ◽  
Jinsai Shang ◽  
Richard Brust ◽  
Jie Zheng ◽  
Patrick R. Griffin ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Activation Function ◽  
Structural Basis ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Exchange Mass Spectrometry ◽  
Pparγ Agonist ◽  
A Cell ◽  
And Function

ABSTRACTThe thiazolidinedione (TZD) pioglitazone (Pio) is an FDA-approved drug for type 2 diabetes mellitus that binds and activates the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ). Although TZDs have potent antidiabetic effects, they also display harmful side effects that have necessitated a better understanding of their mechanisms of action. In particular, little is known about the effect of in vivo TZD metabolites on the structure and function of PPARγ. Here, we present a structure-function comparison of Pio and a major in vivo metabolite, 1-hydroxypioglitazone (PioOH). PioOH displayed a lower binding affinity and reduced potency in coregulator recruitment assays compared to Pio. To determine the structural basis of these findings, we solved an X-ray crystal structure of PioOH bound to PPARγ ligand-binding domain (LBD) and compared it to a published Pio-bound crystal structure. PioOH exhibited an altered hydrogen bonding network that could underlie its reduced affinity and potency compared to Pio. Solution-state structural analysis using NMR spectroscopy and hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis revealed that PioOH stabilizes the PPARγ activation function-2 (AF-2) coactivator binding surface better than Pio. In support of AF-2 stabilization, PioOH displayed stabilized coactivator binding in biochemical assays and better transcriptional efficacy (maximal transactivation response) in a cell-based assay that reports on the activity of the PPARγ LBD. These results, which indicate that Pio hydroxylation affects both its potency and efficacy as a PPARγ agonist, contribute to our understanding of PPARγ-binding drug metabolite interactions and may assist in future PPARγ drug design efforts.

scholarly journals Designed for life: biocompatible de novo designed proteins and components

2018 ◽  
Vol 15 (145) ◽  
pp. 20180472 ◽  
Author(s):  
Katie J. Grayson ◽  
J. L. Ross Anderson
Keyword(s):  
Protein Design ◽  
De Novo ◽  
Building Blocks ◽  
De Novo Design ◽  
Biochemical Processes ◽  
Designed Proteins ◽  
De Novo Protein Design ◽  
Protein Molecules ◽  
And Function

A principal goal of synthetic biology is the de novo design or redesign of biomolecular components. In addition to revealing fundamentally important information regarding natural biomolecular engineering and biochemistry, functional building blocks will ultimately be provided for applications including the manufacture of valuable products and therapeutics. To fully realize this ambitious goal, the designed components must be biocompatible, working in concert with natural biochemical processes and pathways, while not adversely affecting cellular function. For example, de novo protein design has provided us with a wide repertoire of structures and functions, including those that can be assembled and function in vivo . Here we discuss such biocompatible designs, as well as others that have the potential to become biocompatible, including non-protein molecules, and routes to achieving full biological integration.

scholarly journals Neuroimmunoendocrine Regulation of the Prion Protein in Neutrophils

2012 ◽  
Vol 287 (42) ◽  
pp. 35506-35515 ◽  
Author(s):  
Rafael M. Mariante ◽  
Alberto Nóbrega ◽  
Rodrigo A. P. Martins ◽  
Rômulo B. Areal ◽  
Maria Bellio ◽  
...  
Keyword(s):  
Prion Protein ◽  
Surface Protein ◽  
Transmissible Spongiform Encephalopathies ◽  
Spongiform Encephalopathies ◽  
Systemic Injection ◽  
A Cell ◽  
And Function ◽  
Acute And Chronic Inflammation ◽  
The Impact

The prion protein (PrPC) is a cell surface protein expressed mainly in the nervous system. In addition to the role of its abnormal conformer in transmissible spongiform encephalopathies, normal PrPC may be implicated in other degenerative conditions often associated with inflammation. PrPC is also present in cells of hematopoietic origin, including T cells, dendritic cells, and macrophages, and it has been shown to modulate their functions. Here, we investigated the impact of inflammation and stress on the expression and function of PrPC in neutrophils, a cell type critically involved in both acute and chronic inflammation. We found that systemic injection of LPS induced transcription and translation of PrPC in mouse neutrophils. Up-regulation of PrPC was dependent on the serum content of TGF-β and glucocorticoids (GC), which, in turn, are contingent on the activation of the hypothalamic-pituitary-adrenal axis in response to systemic inflammation. GC and TGF-β, either alone or in combination, directly up-regulated PrPC in neutrophils, and accordingly, the blockade of GC receptors in vivo curtailed the LPS-induced increase in the content of PrPC. Moreover, GC also mediated up-regulation of PrPC in neutrophils following noninflammatory restraint stress. Finally, neutrophils with up-regulated PrPC presented enhanced peroxide-dependent cytotoxicity to endothelial cells. The data demonstrate a novel interplay of the nervous, endocrine, and immune systems upon both the expression and function of PrPC in neutrophils, which may have a broad impact upon the physiology and pathology of various organs and systems.

scholarly journals Densified Collagen Tubular Grafts for Human Tissue Replacement and Disease Modelling Applications

2021 ◽  
Author(s):  
Alexander W. Justin ◽  
Sebastian Burgess ◽  
John Ong ◽  
Aishwarya G. Jacob ◽  
Sanjay Sinha ◽  
...  
Keyword(s):  
De Novo ◽  
Disease Modelling ◽  
Luminal Surface ◽  
Luminal Diameter ◽  
Collagen Hydrogel ◽  
Tissue Replacement ◽  
Distinct Cell ◽  
Acid Solubility ◽  
And Function

ABSTRACTFabrication of tubular grafts de novo has been limited by the ability to produce constructs which fulfil the mechanical and biological requirements for implantation and function. In this work, we present a novel method for the formation of densified collagen hydrogel tubular grafts on the scale of human-sized vessels, with the required mechanical strength for future in vivo implantation. The seamless, densified collagen tubes are highly customisable in terms of density, luminal diameter and wall thickness; here we report tubes with luminal diameters 5 mm, 2 mm, and 50 μm, with wall thicknesses of 0.5-3 mm. We show that through genipin crosslinking, acid solubility and swelling of the collagen can be eliminated. Tensile testing shows that axial strength increases with starting collagen and crosslinker concentrations. The cell-compatible densification method enables a high density and uniformly distributed population of cells to be incorporated into the walls of the construct, as well as onto the luminal surface. Additionally, we report a method for generating tubes consisting of distinct cell domains in the walls. The cellular configurations at the boundary between the cell populations may be useful for disease modelling applications. We also demonstrate a method for luminal surface patterning of collagen tubes.

scholarly journals Development, Structure, and Mechanism of Synthetic Antibodies that Target Claudin and Clostridium perfringens Enterotoxin Complexes

2022 ◽  
Author(s):  
Benjamin J. Orlando ◽  
Pawel K. Dominik ◽  
Sourav Roy ◽  
Chinemerem Ogbu ◽  
Satchal K. Erramilli ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Protein Complexes ◽  
Gastrointestinal Diseases ◽  
Phage Display Library ◽  
Surface Receptors ◽  
Terminal Domain ◽  
A Cell ◽  
And Function ◽  
Development Structure

Strains of the Gram-positive bacterium Clostridium perfringens produce a two-domain enterotoxin (CpE) that afflict millions of humans and domesticated animals annually by causing prevalent gastrointestinal illnesses. CpEs C-terminal domain (cCpE) binds cell surface receptors then its N-terminal domain restructures to form a membrane-penetrating 𝛽-barrel pore, which is toxic to epithelial cells of the gut. The claudin family of membrane proteins are the receptors for CpE, and also control the architecture and function of cell/cell contacts called tight junctions that create barriers to intercellular transport of solutes. CpE binding disables claudin and tight junction assembly and induces cytotoxicity via 𝛽-pore formation, disrupting gut homeostasis. Here, we aimed to develop probes of claudin/CpE assembly using a phage display library encoding synthetic antigen-binding fragments (sFabs) and discovered two that bound complexes between human claudin-4 and cCpE. We established each sFabs unique modes of molecular recognition, their binding affinities and kinetics, and determined structures for each sFab bound to ~35 kDa claudin-4/cCpE in three-protein comprised complexes using cryogenic electron microscopy (cryoEM). The structures reveal a recognition epitope common to both sFabs but also that each sFab distinctly conforms to bind their antigen, which explain their unique binding equilibria. Mutagenesis of antigen/sFab interfaces observed therein result in further binding changes. Together, these findings validate the structures and uncover the mechanism of targeting claudin-4/cCpE complexes by these sFabs. Based on these structural insights we generate a model for CpEs cytotoxic claudin-bound 𝛽-pore that predicted that these two sFabs would not prevent CpE cytotoxicity, which we verify in vivo with a cell-based assay. This work demonstrates the development and targeting mechanisms of sFabs against claudin/cCpE that enable rapid structural elucidation of these small membrane protein complexes using a cryoEM workflow. It further provides a structure-based framework and therapeutic strategies for utilizing these sFabs as molecular templates to target claudin/CpE assemblies, obstruct CpE cytotoxicity, and treat CpE-linked gastrointestinal diseases that cause substantial economic and quality of life losses throughout the world.

scholarly journals MED1 is a lipogenesis coactivator required for postnatal adipose expansion

2020 ◽  
Author(s):  
Younghoon Jang ◽  
Young-Kwon Park ◽  
Ji-Eun Lee ◽  
Nhien Tran ◽  
Oksana Gavrilova ◽  
...  
Keyword(s):  
Transcription Factors ◽  
De Novo ◽  
Late Phase ◽  
High Fat ◽  
Maternal Milk ◽  
A Cell ◽  
Coactivator Complex

MED1 often serves as a surrogate of the general transcription coactivator complex Mediator for identifying active enhancers. MED1 is required for phenotypic conversion of fibroblasts to adipocytes in vitro but its role in adipose development and expansion in vivo has not been reported. Here we report that MED1 is dispensable for adipose development in mice. Instead, MED1 is required for postnatal adipose expansion and the induction of de novo lipogenesis (DNL) genes after pups switch diet from high-fat maternal milk to carbohydrate-based chow. During adipogenesis, MED1 is dispensable for induction of lineage-determining transcription factors (TFs) PPARγ and C/EBPα but is required for lipid accumulation in the late phase of differentiation. Mechanistically, MED1 controls the induction of DNL genes by facilitating lipogenic TF ChREBP-dependent recruitment of Mediator to active enhancers. Together, our findings identify a cell- and gene-specific regulatory role of MED1 as a lipogenesis coactivator required for postnatal adipose expansion.

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