A Murine Model of X-Linked Moesin-Associated Immunodeficiency (X-MAID) Reveals Defects in T Cell Homeostasis and Migration

X-linked moesin associated immunodeficiency (X-MAID) is a primary immunodeficiency disease in which patients suffer from profound lymphopenia leading to recurrent infections. The disease is caused by a single point mutation leading to a R171W amino acid change in the protein moesin (moesinR171W). Moesin is a member of the ERM family of proteins, which reversibly link the cortical actin cytoskeleton to the plasma membrane. Here, we describe a novel mouse model with global expression of moesinR171W that recapitulates multiple facets of patient disease, including severe lymphopenia. Further analysis reveals that these mice have diminished numbers of thymocytes and bone marrow precursors. X-MAID mice also exhibit systemic inflammation that is ameliorated by elimination of mature lymphocytes through breeding to a Rag1-deficient background. The few T cells in the periphery of X-MAID mice are highly activated and have mostly lost moesinR171W expression. In contrast, single-positive (SP) thymocytes do not appear activated and retain high expression levels of moesinR171W. Analysis of ex vivo CD4 SP thymocytes reveals defects in chemotactic responses and reduced migration on integrin ligands. While chemokine signaling appears intact, CD4 SP thymocytes from X-MAID mice are unable to polarize and rearrange cytoskeletal elements. This mouse model will be a valuable tool for teasing apart the complexity of the immunodeficiency caused by moesinR171W, and will provide new insights into how the actin cortex regulates lymphocyte function.

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Seeded fibrils of the germline variant of human λ-III immunoglobulin light chain FOR005 have a similar core as patient fibrils with reduced stability

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.016006 ◽

2020 ◽

Vol 295 (52) ◽

pp. 18474-18484

Author(s):

Tejaswini Pradhan ◽

Karthikeyan Annamalai ◽

Riddhiman Sarkar ◽

Stefanie Huhn ◽

Ute Hegenbart ◽

...

Keyword(s):

Solid State ◽

Light Chain ◽

Solid State Nmr ◽

Amyloid Fibrils ◽

Ex Vivo ◽

Chemical Shifts ◽

Single Point ◽

Single Point Mutation ◽

Al Amyloidosis ◽

Fibril Structure

Systemic antibody light chains (AL) amyloidosis is characterized by deposition of amyloid fibrils derived from a particular antibody light chain. Cardiac involvement is a major risk factor for mortality. Using MAS solid-state NMR, we studied the fibril structure of a recombinant light chain fragment corresponding to the fibril protein from patient FOR005, together with fibrils formed by protein sequence variants that are derived from the closest germline (GL) sequence. Both analyzed fibril structures were seeded with ex-vivo amyloid fibrils purified from the explanted heart of this patient. We find that residues 11-42 and 69-102 adopt β-sheet conformation in patient protein fibrils. We identify arginine-49 as a key residue that forms a salt bridge to aspartate-25 in the patient protein fibril structure. In the germline sequence, this residue is replaced by a glycine. Fibrils from the GL protein and from the patient protein harboring the single point mutation R49G can be both heterologously seeded using patient ex-vivo fibrils. Seeded R49G fibrils show an increased heterogeneity in the C-terminal residues 80-102, which is reflected by the disappearance of all resonances of these residues. By contrast, residues 11-42 and 69-77, which are visible in the MAS solid-state NMR spectra, show 13Cα chemical shifts that are highly like patient fibrils. The mutation R49G thus induces a conformational heterogeneity at the C terminus in the fibril state, whereas the overall fibril topology is retained. These findings imply that patient mutations in FOR005 can stabilize the fibril structure.

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Recurrent de novo single point mutation on the gene encoding Na+/K+ pump results in epilepsy

10.1101/2021.08.26.457802 ◽

2021 ◽

Author(s):

Hong-Ming Li ◽

Wen-Bao Hu ◽

Chun-Gu Hong ◽

Ran Duan ◽

Meng-Lu Chen ◽

...

Keyword(s):

Mouse Model ◽

Point Mutation ◽

De Novo ◽

Memory Function ◽

Single Point ◽

Idiopathic Epilepsy ◽

Single Point Mutation ◽

Gene Encoding ◽

Binding Function ◽

Potassium Binding

AbstractThe etiology of epilepsy remains undefined in two-thirds of patients. Here, we identified a de novo mutation of ATP1A2 (c.2426 T>G, p.Leu809Arg), which encodes the α2 subunit of Na+/K+-ATPase, from a family with idiopathic epilepsy. This mutation caused seizures in the study patients. We generated the point mutation mouse model Atp1a2L809R, which recapitulated the epilepsy observed in the study patients. In Atp1a2L809R/WT mice, convulsions were observed and cognitive and memory function was impaired. This mutation affected the potassium binding function of the protein, disabling its ion transport ability, thereby increasing the frequency of nerve impulses. Our work revealed that ATP1A2L809R mutations cause a predisposition to epilepsy. Moreover, we first provide a point mutation mouse model for epilepsy research and drug screening.

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Talin-KANK1 interaction controls the recruitment of cortical microtubule stabilizing complexes to focal adhesions

eLife ◽

10.7554/elife.18124 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 78

Author(s):

Benjamin P Bouchet ◽

Rosemarie E Gough ◽

York-Christoph Ammon ◽

Dieudonnée van de Willige ◽

Harm Post ◽

...

Keyword(s):

Single Point ◽

Cortical Microtubule ◽

Focal Adhesions ◽

Adaptor Protein ◽

Underlying Mechanism ◽

Single Point Mutation ◽

Macromolecular Assemblies ◽

Microtubule Stabilization ◽

Adhesion Sites ◽

And Migration

The cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration. Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote the cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown. Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5β and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin. Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7. Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule-stabilizing complexes with focal adhesions. We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules.

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Selenium Deficiency in a Mouse Model of Sickle Cell Disease Resulted in Increased Oxygen Consumption and Aberrant Mitochondrial Retention (OR11-05-19)

Current Developments in Nutrition ◽

10.1093/cdn/nzz044.or11-05-19 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

Author(s):

Lenny Hong ◽

Ramasamy Jagadeeswaran ◽

Robert Molokie ◽

Donald Lavelle ◽

Angela Rivers ◽

...

Keyword(s):

Oxygen Consumption ◽

Sickle Cell Disease ◽

Mouse Model ◽

Sickle Cell ◽

Globin Gene ◽

Single Point ◽

Selenium Deficiency ◽

Single Point Mutation ◽

Cell Disease ◽

Deficient Diet

Abstract Objectives Sickle Cell Disease (SCD) is caused by a single point mutation in the β-globin gene, resulting in the polymerization of the altered hemoglobin βS in hypoxic conditions, affecting millions of people worldwide. Previous studies have shown that there are lower selenium levels and reduced activity of the antioxidant selenoprotein GPX1 in SCD patients. The objective of this study was to investigate the consequences of selenium deficiency in a SCD mouse model. Methods Humanized SCD (HbSS) mice (Townes model) and wild type (HbAA) mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were fed either a selenium-deficient (<0.01 mg/kg) or a selenium adequate (0.1 mg/kg) diet for 4 weeks. Hematological testing was performed using the ADVIATM 120 analyzer (Bayer Corporation, NY). Mitochondrial retention and reactive oxygen species (ROS) were measured by flow cytometry with a BD LSRFortessaTM analyzer using Kaluza analysis software (Beckman Coulter, CA). The oxygen consumption rate (OCR) was measured from isolated red blood cells (RBCs) in real time using the Seahorse Extracellular Analyzer (Agilent, CA). Results RBCs normally eject their mitochondria before reaching maturity. However, a previous study demonstrated that there was increased RBC mitochondrial retention in SCD mice and patients when compared to controls. Feeding SCD mice a selenium deficient diet resulted in increased retention of mitochondria in RBCs (26% + 6.9%, 5% + 3.5%, n = 3, P < 0.01), decreased hemoglobin levels (5.7 + 0.17 g/dl, 7.0 + 0.83 g/dl, n = 3, P < 0.05), and an increased OCR of the RBCs (P < 0.01) in these animals when compared to SCD mice fed a selenium adequate diet. Conclusions Providing humanized SCD mice a selenium deficient diet resulted in increased mitochondrial retention in mature RBCs, decrease hemoglobin levels, and increased RBC oxygen consumption. RBC retention of mitochondria is associated with increased ROS and hemolysis, potentially contributing to the pain and vaso-occlusive crises that occur in the disease. These studies indicate that selenium deficiency may contribute to the severity of symptoms experienced by patients with SCD. Funding Sources This work is supported by a grant from the NIH.

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Talin-KANK1 interaction controls the recruitment of cortical microtubule stabilizing complexes to focal adhesions

10.1101/055210 ◽

2016 ◽

Cited By ~ 1

Author(s):

Benjamin P. Bouchet ◽

Rosemarie E. Gough ◽

Dieudonnée van de Willige ◽

York-Christoph Ammon ◽

Harm Post ◽

...

Keyword(s):

Single Point ◽

Cortical Microtubule ◽

Focal Adhesions ◽

Adaptor Protein ◽

Underlying Mechanism ◽

Single Point Mutation ◽

Macromolecular Assemblies ◽

Microtubule Stabilization ◽

Adhesion Sites ◽

And Migration

AbstractThe cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration. Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown. Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5P and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin. Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7. Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule-stabilizing complexes with focal adhesions. We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules.

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Amyloidogenic core of a human λ-III immunoglobulin light chain fibril and their germline variants probed by MAS solid state NMR

10.1101/2020.10.02.323303 ◽

2020 ◽

Cited By ~ 1

Author(s):

Tejaswini Pradhan ◽

Karthikeyan Annamalai ◽

Riddhiman Sarkar ◽

Stephanie Huhn ◽

Ute Hegenbart ◽

...

Keyword(s):

Solid State ◽

Light Chain ◽

Solid State Nmr ◽

Amyloid Fibrils ◽

Ex Vivo ◽

Chemical Shifts ◽

Single Point ◽

Single Point Mutation ◽

Al Amyloidosis ◽

Fibril Structure

AbstractSystemic antibody light chains (AL) amyloidosis is characterized by deposition of amyloid fibrils derived from a particular antibody light chain. Cardiac involvement is a major risk factor for mortality. Using MAS solid-state NMR, we study the fibril structure of a recombinant light chain fragment corresponding to the fibril protein from patient FOR005, together with fibrils formed by protein sequence variants that reflect the closest germline (GL) sequence. Both analyzed fibril structures were seeded with ex-vivo amyloid fibrils purified from the explanted heart of this patient. We find that residues 11-42 and 69-102 adopt β-sheet conformation in patient protein fibrils. We identify glycine-49 that is mutated with respect to the germline sequence into arginine-49 as a key residue that forms a salt bridge to aspartate-25 in the patient protein fibril structure. Fibrils from the GL protein and from the patient protein harboring the single point mutation R49G can be both heterologously seeded using patient ex-vivo fibrils. Seeded R49G fibrils show an increased heterogeneity for the C-terminal residues 80-102 which is reflected by the disappearance of all resonances of these residues. By contrast, residues 11-42 and 69-77, which are visible in the MAS solid-state NMR spectra show 13Cα chemical shifts that are highly similar to patient fibrils. The mutation R49G thus induces a conformational heterogeneity at the C-terminus in the fibril state, while the overall fibril topology is retained.

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