Congenital macrothrombocytopenia‐linked mutations in the actin‐binding domain of α‐actinin‐1 enhance F‐actin association

Murphy, Anita C.H.; Lindsay, Andrew J.; McCaffrey, Mary W.; Djinović-Carugo, Kristina; Young, Paul

doi:10.1002/1873-3468.12101

Cited by 30 publications

(30 citation statements)

References 41 publications

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“…ACTN1 is a bundling protein that anchors actin to a variety of intracellular structures. Recently, Murphy et al () have reported that several disease‐associated variants located within the AB domain (p.(Arg46Gln), p.(Val105Ile), and p.(Glu225Lys)) cause increased binding of actinin‐1 to actin filaments. We hypothesize that ACTN1 variants induce more stable actin filaments, thereby impairing stretching and leading to shorter and thicker fibers in transfected CHO cells.…”

Section: Discussionmentioning

confidence: 99%

“…ACTN1 is an antiparallel, dimeric, actin cross‐linking protein composed of three domains: the N‐terminal actin‐binding (AB) domain composed of two calponin homology domains; the C‐terminal calmodulin (CaM) domain, which binds another actinin molecule, and the central rod domain composed of four spectrin‐like repeats (SRs) (Gimona & Mital, ; Murphy, Lindsay, McCaffrey, Djinović‐Carugo, & Young, ; Sjöblom, Salmazo, & Djinović‐Carugo, ). Twenty‐five ACTN1 variants have been described so far, of which 16 have been shown to alter the AB and CaM domains (Bottega et al, ; Faleschini et al, ; Guéguen et al, ; Kunishima et al, ) and induce varying degrees of actin network disorganization, while four variants were localized in the neck domains flanking the rod domain (Bottega et al, ; Kunishima et al, ).…”

Section: Introductionmentioning

confidence: 99%

“…Twenty‐five ACTN1 variants have been described so far, of which 16 have been shown to alter the AB and CaM domains (Bottega et al, ; Faleschini et al, ; Guéguen et al, ; Kunishima et al, ) and induce varying degrees of actin network disorganization, while four variants were localized in the neck domains flanking the rod domain (Bottega et al, ; Kunishima et al, ). Variants in the AB domain have been demonstrated to induce increased ACTN1 binding to actin filaments (Murphy et al, ). However, functional data on variants affecting the rod domain are scarce.…”

Section: Introductionmentioning

confidence: 99%

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NovelACTN1variants in cases of thrombocytopenia

Vincenot¹,

Saultier

Kunishima

et al. 2019

Human Mutation

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The ACTN1 gene has been implicated in inherited macrothrombocytopenia. To decipher the spectrum of variants and phenotype of ACTN1‐related thrombocytopenia, we sequenced the ACTN1 gene in 272 cases of unexplained chronic or familial thrombocytopenia. We identified 15 rare, monoallelic, nonsynonymous and likely pathogenic ACTN1 variants in 20 index cases from 20 unrelated families. Thirty‐one family members exhibited thrombocytopenia. Targeted sequencing was carried out on 12 affected relatives, which confirmed presence of the variant. Twenty‐eight of 32 cases with monoallelic ACTN1 variants had mild to no bleeding complications. Eleven cases harbored 11 different unreported ACTN1 variants that were monoallelic and likely pathogenic. Nine variants were located in the α‐actinin‐1 (ACTN1) rod domain and were predicted to hinder dimer formation. These variants displayed a smaller increase in platelet size compared with variants located outside the rod domain. In vitro expression of the new ACTN1 variants induced actin network disorganization and led to increased thickness of actin fibers. These findings expand the repertoire of ACTN1 variants associated with thrombocytopenia and highlight the high frequency of ACTN1‐related thrombocytopenia cases. The rod domain, like other ACTN1 functional domains, may be mutated resulting in actin disorganization in vitro and thrombocytopenia with normal platelet size in most cases.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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NovelACTN1variants in cases of thrombocytopenia

Vincenot¹,

Saultier

Kunishima

et al. 2019

Human Mutation

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“…Given that the actin binding and the CH domains are responsible for interaction with actin, alterations of α‐actinin 1 are likely to impair the actin filament organization. It has been recently reported that some of these mutant forms exert a gain‐of‐function effect, resulting in an increased binding to actin, which is likely to affect its filament dynamics (Murphy et al , ). Indeed, the cytoskeleton structure is defective in cells expressing 15 out of the 26 variants identified in the ACTN1 gene, including the two novel mutations p.Trp128Cys and p.Pro233Leu of this paper (Fig ) (Kunishima et al , ; Bottega et al , ; Yasutomi et al , ).…”

Section: Discussionmentioning

confidence: 99%

ACTN1 mutations lead to a benign form of platelet macrocytosis not always associated with thrombocytopenia

et al. 2018

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M.F. and F.M. contributed equally to this study. SummaryThe inherited thrombocytopenias (IT) are a heterogeneous group of diseases resulting from mutations in more than 30 different genes. Among them, ACTN1-related thrombocytopenia (ACTN1-RT; Online Mendelian Inheritance in Man: 615193) is one of the most recently identified forms. It has been described as a mild autosomal dominant macrothrombocytopenia caused by mutations in ACTN1, a gene encoding for one of the two nonmuscle isoforms of a-actinin. We recently identified seven new unrelated families with ACTN1-RT caused by different mutations. Two of them are novel missense variants (p.Trp128Cys and p.Pro233Leu), whose pathogenic role has been confirmed by in vitro studies. Together with the 10 families we have previously described, our cohort of ACTN1-RT now consists of 49 individuals carrying ACTN1 mutations. This is the largest case series ever collected and enabled a critical evaluation of the clinical aspects of the disease. We concluded that ACTN1-RT is the fourth most frequent form of IT worldwide and it is characterized by platelet macrocytosis in all affected subjects and mild thrombocytopenia in less than 80% of cases. The risk of bleeding, either spontaneous or upon haemostatic challenge, is negligible and there are no other associated defects, either congenital or acquired. Therefore, ACTN1-RT is a benign form of IT, whose diagnosis provides affected individuals and their families with a good prognosis.

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“…Cells were incubated in blocking buffer (0.1% triton X-100, 5% goat serum and 2% bovine serum in PBS) prior to antibody incubation. All antibodies were diluted in 5% goat serum and 2% bovine serum in PBS (as described [ 20 ]). Cells were washed three times in PBS following each antibody incubation and coverslips were mounted onto glass slides using Fluoromount mounting media.…”

Section: Methodsmentioning

confidence: 99%

Proteomic analysis reveals novel ligands and substrates for LNX1 E3 ubiquitin ligase

2017

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Ligand of Numb protein X1 (LNX1) is an E3 ubiquitin ligase that contains a catalytic RING (Really Interesting New Gene) domain and four PDZ (PSD-95, DlgA, ZO-1) domains. LNX1 can ubiquitinate Numb, as well as a number of other ligands. However, the physiological relevance of these interactions in vivo remain unclear. To gain functional insights into the LNX family, we have characterised the LNX1 interactome using affinity purification and mass spectrometry. This approach identified a large number of novel LNX1-interacting proteins, as well as confirming known interactions with NUMB and ERC2. Many of the novel interactions mapped to the LNX PDZ domains, particularly PDZ2, and many showed specificity for LNX1 over the closely related LNX2. We show that PPFIA1 (liprin-α1), KLHL11, KIF7 and ERC2 are substrates for ubiquitination by LNX1. LNX1 ubiquitination of liprin-α1 is dependent on a PDZ binding motif containing a carboxyl terminal cysteine that binds LNX1 PDZ2. Surprisingly, the neuronally-expressed LNX1p70 isoform, that lacks the RING domain, was found to promote ubiquitination of PPFIA1 and KLHL11, albeit to a lesser extent than the longer RING-containing LNX1p80 isoform. Of several E3-ligases identified in the LNX1 interactome we confirm interactions of LNX1 with MID2/TRIM1 and TRIM27. On this basis we propose a model whereby LNX1p70, despite lacking a catalytic RING domain, may function as a scaffold to promote ubiquitination of its ligands through recruitment of other E3-ligases. These findings provide functional insights into the LNX protein family, particularly the neuronal LNX1p70 isoform.

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Congenital macrothrombocytopenia‐linked mutations in the actin‐binding domain of α‐actinin‐1 enhance F‐actin association

Cited by 30 publications

References 41 publications

NovelACTN1variants in cases of thrombocytopenia

NovelACTN1variants in cases of thrombocytopenia

ACTN1 mutations lead to a benign form of platelet macrocytosis not always associated with thrombocytopenia

Proteomic analysis reveals novel ligands and substrates for LNX1 E3 ubiquitin ligase

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