scholarly journals Multiple Sclerosis-Associated hnRNPA1 Mutations Alter hnRNPA1 Dynamics and Influence Stress Granule Formation

2021 ◽  
Vol 22 (6) ◽  
pp. 2909
Author(s):  
Joseph-Patrick W. E. Clarke ◽  
Patricia A. Thibault ◽  
Hannah E. Salapa ◽  
David E. Kim ◽  
Catherine Hutchinson ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Blue Light ◽  
Molecular Mechanisms ◽  
Imaging System ◽  
Cluster Formation ◽  
Expression System ◽  
Stress Granule ◽  
Granule Formation ◽  
Cryptochrome 2

Evidence indicates that dysfunctional heterogeneous ribonucleoprotein A1 (hnRNPA1; A1) contributes to the pathogenesis of neurodegeneration in multiple sclerosis. Understanding molecular mechanisms of neurodegeneration in multiple sclerosis may result in novel therapies that attenuate neurodegeneration, thereby improving the lives of MS patients with multiple sclerosis. Using an in vitro, blue light induced, optogenetic protein expression system containing the optogene Cryptochrome 2 and a fluorescent mCherry reporter, we examined the effects of multiple sclerosis-associated somatic A1 mutations (P275S and F281L) in A1 localization, cluster kinetics and stress granule formation in real-time. We show that A1 mutations caused cytoplasmic mislocalization, and significantly altered the kinetics of A1 cluster formation/dissociation, and the quantity and size of clusters. A1 mutations also caused stress granule formation to occur more quickly and frequently in response to blue light stimulation. This study establishes a live cell optogenetics imaging system to probe localization and association characteristics of A1. It also demonstrates that somatic mutations in A1 alter its function and promote stress granule formation, which supports the hypothesis that A1 dysfunction may exacerbate neurodegeneration in multiple sclerosis.

scholarly journals Sorting and storage during secretory granule biogenesis: looking backward and looking forward

1998 ◽  
Vol 332 (3) ◽  
pp. 593-610 ◽  
Author(s):  
Peter ARVAN ◽  
David CASTLE
Keyword(s):  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Secretory Granules ◽  
Secretory Proteins ◽  
Membrane Composition ◽  
Granule Formation ◽  
Granule Membrane ◽  
Regulated Secretory Pathway ◽  
Secretory Products

Secretory granules are specialized intracellular organelles that serve as a storage pool for selected secretory products. The exocytosis of secretory granules is markedly amplified under physiologically stimulated conditions. While granules have been recognized as post-Golgi carriers for almost 40 years, the molecular mechanisms involved in their formation from the trans-Golgi network are only beginning to be defined. This review summarizes and evaluates current information about how secretory proteins are thought to be sorted for the regulated secretory pathway and how these activities are positioned with respect to other post-Golgi sorting events that must occur in parallel. In the first half of the review, the emerging role of immature secretory granules in protein sorting is highlighted. The second half of the review summarizes what is known about the composition of granule membranes. The numerous similarities and relatively limited differences identified between granule membranes and other vesicular carriers that convey products to and from the plasmalemma, serve as a basis for examining how granule membrane composition might be established and how its unique functions interface with general post-Golgi membrane traffic. Studies of granule formation in vitro offer additional new insights, but also important challenges for future efforts to understand how regulated secretory pathways are constructed and maintained.

scholarly journals Cross-talk of anosmin-1, the protein implicated in X-linked Kallmann's syndrome, with heparan sulphate and urokinase-type plasminogen activator

2004 ◽  
Vol 384 (3) ◽  
pp. 495-505 ◽  
Author(s):  
Youli HU ◽  
David GONZÁLEZ-MARTÍNEZ ◽  
Soo-Hyun KIM ◽  
Pierre Marc Gilles BOULOUX
Keyword(s):  
Cell Surface ◽  
Plasminogen Activator ◽  
Molecular Mechanisms ◽  
Expression System ◽  
Heparan Sulphate ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Kallmann's Syndrome ◽  
Kallmann’S Syndrome

Defective function of anosmin-1, the protein encoded by KAL-1, underlies X-linked Kallmann's syndrome (X-KS), a human hereditary developmental disorder. Anosmin-1 appears to play a role in neurite outgrowth and axon branching, although molecular mechanisms of its action are still unknown. Anosmin-1 contains a WAP (whey acidic protein-like) domain and four contiguous FnIII (fibronectin-like type III) repeats; its WAP domain shows similarity to known serine protease inhibitors, whereas the FnIII domains contain HS (heparan sulphate)-binding sequences. To investigate the functional role of these domains, we have generated both wild-type and mutant recombinant anosmin-1 proteins using a Drosophila S2 cell expression system. Here we present the first biochemical evidence demonstrating the high-binding affinity between HS and anosmin-1, as measured by SPR (surface plasmon resonance) (Kd=2 nM). The FnIII domains, particularly the first, are essential for dose-dependent HS binding and HS-mediated cell surface association. Furthermore, we have identified uPA (urokinase-type plasminogen activator) as an anosmin-1 interactant. Anosmin-1 significantly enhances the amidolytic activity of uPA in vitro; and anosmin-1–HS–uPA co-operation induces cell proliferation in the PC-3 prostate carcinoma cell line. Both the HS interaction and an intact WAP domain are required for the mitogenic activity of anosmin-1. These effects appear to be mediated by a direct protein interaction between anosmin-1 and uPA, since anosmin-1–uPA could be co-immunoprecipitated from PC-3 cell lysates, and their direct binding with high affinity (Kd=6.91 nM) was demonstrated by SPR. We thus propose that anosmin-1 may modulate the catalytic activity of uPA and its signalling pathway, whereas HS determines cell surface localization of the anosmin-1–uPA complex.

scholarly journals Cells Lacking the Fragile X Mental Retardation Protein (FMRP) have Normal RISC Activity but Exhibit Altered Stress Granule Assembly

2009 ◽  
Vol 20 (1) ◽  
pp. 428-437 ◽  
Author(s):  
Marie-Cécile Didiot ◽  
Murugan Subramanian ◽  
Eric Flatter ◽  
Jean-Louis Mandel ◽  
Hervé Moine
Keyword(s):  
Mental Retardation ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Fragile X ◽  
Stress Granule ◽  
Translation Regulation ◽  
Positive Role ◽  
Granule Formation ◽  
Fragile X Mental Retardation ◽  
Mental Retardation Protein

The fragile X mental retardation protein (FMRP) is an RNA-binding protein involved in the mRNA metabolism. The absence of FMRP in neurons leads to alterations of the synaptic plasticity, probably as a result of translation regulation defects. The exact molecular mechanisms by which FMRP plays a role in translation regulation have remained elusive. The finding of an interaction between FMRP and the RNA interference silencing complex (RISC), a master of translation regulation, has suggested that both regulators could be functionally linked. We investigated here this link, and we show that FMRP exhibits little overlap both physically and functionally with the RISC machinery, excluding a direct impact of FMRP on RISC function. Our data indicate that FMRP and RISC are associated to distinct pools of mRNAs. FMRP, unlike RISC machinery, associates with the pool of mRNAs that eventually goes into stress granules upon cellular stress. Furthermore, we show that FMRP plays a positive role in this process as the lack of FMRP or a point mutant causing a severe fragile X alter stress granule formation. Our data support the proposal that FMRP plays a role in controlling the fate of mRNAs after translation arrest.

Functional expression and RNA binding analysis of the interferon-induced, double-stranded RNA-activated, 68,000-Mr protein kinase in a cell-free system

1991 ◽  
Vol 11 (11) ◽  
pp. 5497-5505
Author(s):  
M G Katze ◽  
M Wambach ◽  
M L Wong ◽  
M Garfinkel ◽  
E Meurs ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Expression System ◽  
In Vitro Translation ◽  
Initiation Factor ◽  
Alpha Subunit ◽  
Single Amino Acid ◽  
Free System

Eukaryotic viruses have devised numerous strategies to downregulate activity of the interferon-induced, double-stranded (dsRNA)-activated protein kinase (referred to as p68 on the basis of its Mr of 68,000 in human cells). Viruses must exert this control to avoid extensive phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2) by p68 and the resultant negative effects on protein synthesis initiation. To begin to define the molecular mechanisms underlying this regulation, we optimized expression of p68 in an in vitro transcription-translation system utilizing the full-length cDNA clone. The in vitro-expressed kinase was autophosphorylated in response to dsRNAs and heparin in a manner similar to that for the native p68 provided that the kinase inhibitor, 2-aminopurine, was present during the in vitro translation reaction. Further, the activated kinase efficiently phosphorylated its natural substrate, the alpha subunit of eIF-2. Binding experiments revealed that the expressed kinase complexed with the dsRNA activator, reovirus dsRNA, as well as the adenovirus-encoded inhibitor, VAI RNA. Interestingly, both the reovirus RNAs and VAI RNA also complexed with protein kinase molecules that lacked the carboxyl terminus and all catalytic domains. Deletion analysis confirmed that the p68 amino terminus contained critical determinants for reovirus dsRNA and VAI RNA binding. Further, reovirus dsRNA efficiently bound to, but failed to activate, p68 kinase molecules containing a single amino acid substitution in the invariant lysine 295 present in catalytic domain II. Taken together, these data demonstrate that this expression system permits a detailed mutagenic analysis of the regions of p68 required for interaction with virus-encoded activators and repressors.

scholarly journals 0028 Sleep Duration Influences the Kinetics of Stress Granule Formation

SLEEP ◽  
2020 ◽  
Vol 43 (Supplement_1) ◽  
pp. A11-A12
Author(s):  
M K Dougherty ◽  
C Saul ◽  
L Carman ◽  
M D Nelson ◽  
J C Tudor
Keyword(s):  
Sleep Deprivation ◽  
Sleep Duration ◽  
Protein A ◽  
Stress Granules ◽  
Mtor Pathway ◽  
Stress Granule ◽  
Target Of Rapamycin ◽  
Granule Formation ◽  
One Way Anova

Abstract Introduction Stress granules are non-membrane bound aggregates of messenger ribonucleoproteins that are biomarkers of cellular stress. It has been shown in cells in vitro that suppression of the mammalian target of rapamycin (mTOR) pathway and its non-mammalian orthologue target of rapamycin (TOR) is associated with an increase in stress granule formation. It has also been shown that the mTOR pathway is suppressed in response to sleep deprivation in mice. Despite the possible connection via the TOR/mTOR pathway, there has not been any previous evidence linking sleep deprivation with stress granule formation. Methods Our present investigation uses the nematode Caenorhabditis elegans to model how stress granule formation and clearance are modified by sleep duration. We developed novel strains of C. elegans that model each type of sleep deprivation or enhancement and have RFP-labeled PAB-1 protein, a key component of stress granules. In addition to modifying sleep duration via genetic means, we also sleep deprived wildtype fluorescently labeled animals using mechanical disturbances. Results Animals with enhanced stress-induced sleep have stress granules that are smaller in size and cleared faster than wildtype, while sleep deprived animals have granules that are slower to clear (F11,473 = 7.752, ***p < 0.0001, one-way ANOVA). Animals that were manually deprived of stress-induced sleep were similarly slower to clear stress granules (F5,209 = 5.476 ***p < 0.0001, one-way ANOVA). Interestingly, animals genetically deprived of developmentally-timed sleep does not appear to have more stress granules in the middle of their sleep period than the sleeping wildtype stage (F2,42 = 2.659, p = 0.0729, one-way ANOVA). Conclusion This work demonstrates that the amount of sleep affects stress granule kinetics, which impacts the flow of genetic information inside cells. Support This work was supported by an R15GM122058 (NIH), John P. McNulty scholars program (SJU) and summer scholars program (SJU).

Generation and characterization of U937-TR: a platform cell line for inducible gene expression in human macrophages

Parasitology ◽  
2020 ◽  
Vol 147 (13) ◽  
pp. 1524-1531
Author(s):  
Cristian Camilo Galindo ◽  
Carlos Arturo Clavijo-Ramírez
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Molecular Mechanisms ◽  
Expression System ◽  
Precise Control ◽  
Expression Of Genes ◽  
Wide Range ◽  
Monocytes And Macrophages

AbstractMonocytes and macrophages are involved in a wide range of biological processes and parasitic diseases. The characterization of the molecular mechanisms governing such processes usually requires precise control of the expression of genes of interest. We implemented a tetracycline-controlled gene expression system in the U937 cell line, one of the most used in vitro models for the research of human monocytes and macrophages. Here we characterized U937-derived cell lines in terms of phenotypic (morphology and marker expression) and functional (capacity for phagocytosis and for Leishmania parasite hosting) changes induced by phorbol-12-myristate-13-acetate (PMA). Finally, we provide evidence of tetracycline-inducible and reversible Lamin-A gene silencing of the PMA-differentiated U937-derived cells.

Understanding the Molecular Basis of Cell Migration; Implications for Clinical Therapy in Multiple Sclerosis

1997 ◽  
Vol 92 (2) ◽  
pp. 113-122 ◽  
Author(s):  
Richard Milner
Keyword(s):  
Multiple Sclerosis ◽  
Central Nervous System ◽  
Nervous System ◽  
Cell Migration ◽  
Molecular Mechanisms ◽  
Autoimmune Response ◽  
The Central Nervous System ◽  
Oligodendrocyte Precursor ◽  
Oligodendrocyte Precursors

1. Multiple sclerosis is characterized by areas of demyelination spread throughout the central nervous system, in which the myelin sheaths surrounding axons are destroyed. While therapies aimed at suppressing the autoimmune response, such as β-interferon, may prevent further damage, they cannot repair or replace the lost myelin. To this end, an additional therapy has been proposed, which involves transplanting cells of the oligodendrocyte lineage into the central nervous system. 2. The cell of interest for transplantation is the oligodendrocyte precursor because, unlike the differentiated cell, it is an intrinsically migratory and proliferative cell. In order to optimize the transplant strategy we have investigated the molecular mechanisms that control migration in vitro, so that these mechanisms might be upregulated to maximize cell migration in vivo. We have focused on the integrin family of cell adhesion molecules, known to play a fundamental role in the regulation of migration in other cell types. 3. These studies show that oligodendrocytes express a limited repertoire of integrins consisting of α6β1 and three different αv integrins. α6β1 is expressed throughout development but αv integrins show developmental regulation; differentiation is accompanied by loss of αvβ1 and upregulation of αvβ5. 4. Function-blocking studies show that oligodendrocyte precursor migration in vitro is mediated primarily by the developmentally regulated αvβ1 integrin, but not α6β1 or αvβ3. Taken together with previous evidence that cell migration can be regulated by altering integrin expression, this work suggests that modifying expression levels of αvβ1 on oligodendrocyte precursors may increase the migratory capacity of these cells. If so, this would support a future therapeutic strategy aimed at transplanting genetically modified oligodendrocyte precursors to repair widespread demyelinated lesions.

scholarly journals Functional expression and RNA binding analysis of the interferon-induced, double-stranded RNA-activated, 68,000-Mr protein kinase in a cell-free system.

1991 ◽  
Vol 11 (11) ◽  
pp. 5497-5505 ◽  
Author(s):  
M G Katze ◽  
M Wambach ◽  
M L Wong ◽  
M Garfinkel ◽  
E Meurs ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Expression System ◽  
In Vitro Translation ◽  
Initiation Factor ◽  
Alpha Subunit ◽  
Single Amino Acid ◽  
Free System

Eukaryotic viruses have devised numerous strategies to downregulate activity of the interferon-induced, double-stranded (dsRNA)-activated protein kinase (referred to as p68 on the basis of its Mr of 68,000 in human cells). Viruses must exert this control to avoid extensive phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2) by p68 and the resultant negative effects on protein synthesis initiation. To begin to define the molecular mechanisms underlying this regulation, we optimized expression of p68 in an in vitro transcription-translation system utilizing the full-length cDNA clone. The in vitro-expressed kinase was autophosphorylated in response to dsRNAs and heparin in a manner similar to that for the native p68 provided that the kinase inhibitor, 2-aminopurine, was present during the in vitro translation reaction. Further, the activated kinase efficiently phosphorylated its natural substrate, the alpha subunit of eIF-2. Binding experiments revealed that the expressed kinase complexed with the dsRNA activator, reovirus dsRNA, as well as the adenovirus-encoded inhibitor, VAI RNA. Interestingly, both the reovirus RNAs and VAI RNA also complexed with protein kinase molecules that lacked the carboxyl terminus and all catalytic domains. Deletion analysis confirmed that the p68 amino terminus contained critical determinants for reovirus dsRNA and VAI RNA binding. Further, reovirus dsRNA efficiently bound to, but failed to activate, p68 kinase molecules containing a single amino acid substitution in the invariant lysine 295 present in catalytic domain II. Taken together, these data demonstrate that this expression system permits a detailed mutagenic analysis of the regions of p68 required for interaction with virus-encoded activators and repressors.

scholarly journals Casein Kinase 2 Is Linked to Stress Granule Dynamics through Phosphorylation of the Stress Granule Nucleating Protein G3BP1

2016 ◽  
Vol 37 (4) ◽  
Author(s):  
Lucas C. Reineke ◽  
Wei-Chih Tsai ◽  
Antrix Jain ◽  
Jason T. Kaelber ◽  
Sung Yun Jung ◽  
...  
Keyword(s):  
Cell Fate ◽  
Stress Granules ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Stress Granule ◽  
Translational Repression ◽  
Transcriptional Responses ◽  
Granule Formation ◽  
Cell Fate Decisions

ABSTRACT Stress granules (SGs) are large macromolecular aggregates that contain translation initiation complexes and mRNAs. Stress granule formation coincides with translational repression, and stress granules actively signal to mediate cell fate decisions by signaling to the translation apparatus to (i) maintain translational repression, (ii) mount various transcriptional responses, including innate immunity, and (iii) repress apoptosis. Previous work showed that G3BP1 is phosphorylated at serine 149, which regulates G3BP1 oligomerization, stress granule assembly, and RNase activity intrinsic to G3BP1. However, the kinase that phosphorylates G3BP1 was not identified, leaving a key step in stress granule regulation uncharacterized. Here, using chemical inhibition, genetic depletion, and overexpression experiments, we show that casein kinase 2 (CK2) promotes stress granule dynamics. These results link CK2 activity with SG disassembly. We also show that casein kinase 2 phosphorylates G3BP1 at serine 149 in vitro and in cells. These data support a role for casein kinase 2 in regulation of protein synthesis by downregulating stress granule formation through G3BP1.

scholarly journals Modulation of RNA condensation by the DEAD-box protein eIF4A

2019 ◽  
Author(s):  
Devin Tauber ◽  
Gabriel Tauber ◽  
Anthony Khong ◽  
Briana Van Treeck ◽  
Jerry Pelletier ◽  
...  
Keyword(s):  
Stress Granules ◽  
Protein Aggregates ◽  
Stress Granule ◽  
List Type ◽  
Dependent Manner ◽  
Granule Formation ◽  
Dead Box ◽  
The Dead ◽  
In Cells

SUMMARYStress granules are condensates of non-translating mRNAs and proteins involved in the stress response and neurodegenerative diseases. Stress granules form in part through intermolecular RNA-RNA interactions, although the process of RNA condensation is poorly understood. In vitro, we demonstrate that RNA is effectively recruited to the surfaces of RNA or RNP condensates. We demonstrate that the DEAD-box protein eIF4A reduces RNA condensation in vitro and limits stress granule formation in cells. This defines a purpose for eIF4A to limit intermolecular RNA-RNA interactions in cells, thereby allowing for proper RNP function. These results establish an important role for DEAD-box proteins as ATP-dependent RNA chaperones that can limit the intermolecular condensation and entanglement of RNA, analogous to the function of proteins like HSP70 in combatting protein aggregates.eTOC BlurbStress granules are formed in part by the process of RNA condensation, which is mediated by and promotes trans RNA-RNA interactions. The essential DEAD-box protein and translation initiation factor eIF4A limits stress granule formation by reducing RNA condensation through its function as an ATP-dependent RNA binding protein, behaving analogously to how protein chaperones like HSP70 combat protein aggregates.HighlightsRNA condensates promote intermolecular RNA-RNA interactions at their surfaceseIF4A limits the recruitment of RNAs to stress granules in cellseIF4A reduces the nucleation of stress granules in cellsRecombinant eIF4A1 inhibits the condensation of RNA in vitro in an ATP-dependent manner

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