Functional and Structural Analysis of the Conserved EFhd2 Protein

Ferrer-Acosta, Yancy; Cruz, Eva N. Rodriguez; Vaquer, Ana del C.; Vega, Irving E.

doi:10.2174/0929866511320050011

Cited by 24 publications

(45 citation statements)

References 35 publications

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“…4b). Consistent with a previous study showing that thermostability of EFhd2 was restored by Ca 2+ at a high temperature31, the half aggregation temperature for both EF-hand mutants that bind only one Ca 2+ is significantly lower ( CD EFhd2 EF1 : 62.32 ± 0.14 °C, CD EFhd2 EF2 : 57.90 ± 0.60 °C) than the two Ca 2+ -bound EFhd2 ( CD EFhd2: 84.89 ± 0.01 °C) and is consistent with ref. 30 and 31.…”

Section: Resultssupporting

confidence: 93%

“…Taking into consideration a previous study and structural similarity between Ca 2+ -peptide-CaM complexes, the observations for STIM1 and CD EFhd2 support the hypothesis that high affinity for Ca 2+ and intramolecular interactions of CD EFhd2 are likely to maximise stabilisation of the EFhd2 fold. In support of this hypothesis is the thermostability results of EFhd2, which showed that the protein thermal stability at high temperature was restored by Ca 2+ 31. This is further emphasised by the observation that CD EFhd2 remained stable in solution, even at high temperatures in the presence of two Ca 2+ ions (Fig.…”

Section: Discussionmentioning

confidence: 60%

“…To evaluate structural dynamics at the atomic level, we performed ensemble refinement using the Phenix.ensemble refinement31 for CD EFhd2, CD EFhd2 EF1 and CD EFhd2 EF2 . Harmonic restraints were applied for all amino acids with visible electron density at a level of 1σ in the 2m F o-D F c electron density map using parameters slack = 1.0 and weight = 0.001.…”

Section: Methodsmentioning

confidence: 99%

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Structural implications of Ca2+-dependent actin-bundling function of human EFhd2/Swiprosin-1

Park

Kwon

et al. 2016

Sci Rep

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EFhd2/Swiprosin-1 is a cytoskeletal Ca2+-binding protein implicated in Ca2+-dependent cell spreading and migration in epithelial cells. EFhd2 domain architecture includes an N-terminal disordered region, a PxxP motif, two EF-hands, a ligand mimic helix and a C-terminal coiled-coil domain. We reported previously that EFhd2 displays F-actin bundling activity in the presence of Ca2+ and this activity depends on the coiled-coil domain and direct interaction of the EFhd2 core region. However, the molecular mechanism for the regulation of F-actin binding and bundling by EFhd2 is unknown. Here, the Ca2+-bound crystal structure of the EFhd2 core region is presented and structures of mutants defective for Ca2+-binding are also described. These structures and biochemical analyses reveal that the F-actin bundling activity of EFhd2 depends on the structural rigidity of F-actin binding sites conferred by binding of the EF-hands to Ca2+. In the absence of Ca2+, the EFhd2 core region exhibits local conformational flexibility around the EF-hand domain and C-terminal linker, which retains F-actin binding activity but loses the ability to bundle F-actin. In addition, we establish that dimerisation of EFhd2 via the C-terminal coiled-coil domain, which is necessary for F-actin bundling, occurs through the parallel coiled-coil interaction.

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Section: Resultssupporting

confidence: 93%

Section: Discussionmentioning

confidence: 60%

Section: Methodsmentioning

confidence: 99%

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Structural implications of Ca2+-dependent actin-bundling function of human EFhd2/Swiprosin-1

Park

Kwon

et al. 2016

Sci Rep

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“…To date, no study has linked EFHD2 with PD. Multiple sequence alignment of EFHD2 showed that the identified phosphosite, serine 74, is highly conserved among several species (data not shown) and lies within the N-terminal region of EFHD2 that may be important for regulating calcium-binding activity (Ferrer-Acosta et al , 2013a). FKBP38 is a membrane chaperone predominantly localised in mitochondria.…”

Section: Discussionmentioning

confidence: 99%

Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1

et al. 2015

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Mutations in the PTEN-induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser65) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1-dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub-family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser111) in response to PINK1 activation. Using phospho-specific antibodies raised against Ser111 of each of the Rabs, we demonstrate that Rab Ser111 phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient-derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser111 phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser111 phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser65. We further show mechanistically that phosphorylation at Ser111 significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser111 may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase-mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease.

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“…(1) Both proteins bind Ca 2 + . (2)(3)(4) EFhd2 is highly expressed in the brain, (5) has been proposed to be a calcium sensor protein, (3) and is putatively linked to neurodegenerative diseases (see review (1) ). EFhd1 has been shown to be involved in neuronal differentiation in a cell line model.…”

Section: Introductionmentioning

confidence: 99%

Monoclonal Antibodies to Discriminate the EF Hand Containing Calcium Binding Adaptor Proteins EFhd1 and EFhd2

Brachs

Lang

Buslei

et al. 2013

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

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Small Ca(2+) binding adaptor proteins of the EF hand family play important roles in neuronal and immune cell Ca(2+) signaling. Swiprosin-1/EFhd2 (EFhd2) and Swiprosin-2/EFhd1 (EFhd1) are conserved and very homologous Ca(2+) binding adaptor proteins of the EF hand family, with possibly redundant functions. In particular, EFhd2 has been proposed to be involved in B cell signaling and neuropathological disorders. Little is known thus far about the expression of EFhd2 on the single cell level in tissue sections or blood cells. Here we describe the generation of four specific anti-EFhd2 monoclonal antibodies. These recognize murine and human EFhd2, but not murine EFhd1, and their binding site maps to a region in the N-terminal part of EFhd2, where EFhd2 and EFhd1 differ most. Moreover, to detect EFhd1 specifically, we also generated anti-EFhd1 polyclonal antibodies, making use of a singular peptide of the N-terminal part of the protein. Using anti-EFhd2 MAb, we reveal two EFhd2 pools in B cells, one at the membrane and one cytoplasmic pool. Staining of human peripheral blood mononuclear cells shows EFhd2 expression in B cells but a ∼5 fold higher expression in monocytes. Taken together, EFhd2 monoclonal antibodies will be valuable to assess the real subcellular localization and expression level of EFhd2 in healthy and diseased primary cells and tissues.

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Functional and Structural Analysis of the Conserved EFhd2 Protein

Cited by 24 publications

References 35 publications

Structural implications of Ca2+-dependent actin-bundling function of human EFhd2/Swiprosin-1

Structural implications of Ca2+-dependent actin-bundling function of human EFhd2/Swiprosin-1

Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1

Monoclonal Antibodies to Discriminate the EF Hand Containing Calcium Binding Adaptor Proteins EFhd1 and EFhd2

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