Genotype-Structure-Phenotype Correlations in Disease-Associated IGF1R Variants and Similarities to Those in INSR Variants

We previously reported that genotype-phenotype correlations in 12 missense variants causing severe insulin resistance, located in the second and third fibronectin type III (FnIII) domains of the insulin receptor (INSR), containing the α-β cleavage and part of insulin-binding sites. This study aimed to identify genotype-phenotype correlations in FnIII domain variants of IGF1R, a structurally related homolog of INSR, which may be associated with growth retardation, using the recently reported crystal structures of IGF1R. A structural bioinformatics analysis of five previously reported disease-associated heterozygous missense variants and a likely benign variant in the FnIII domains of IGF1R predicted that the disease-associated variants would severely impair the hydrophobic core formation and stability of the FnIII domains or affect the α-β cleavage site, while the likely benign variant would not affect the folding of the domains. A functional analysis of these variants in CHO cells showed impaired receptor processing and autophosphorylation in cells expressing the disease-associated variants, but not in those expressing the wild-type form or the likely benign variant. These results demonstrated genotype-phenotype correlations in the FnIII domain variants of IGF1R, which are presumably consistent with those of INSR and would help in the early diagnosis of patients with disease-associated IGF1R variants.

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Ligand-specific utilization of the extracellular membrane-proximal region of the gp130-related signalling receptors

Biochemical Journal ◽

10.1042/bj3450025 ◽

1999 ◽

Vol 345 (1) ◽

pp. 25-32 ◽

Cited By ~ 9

Author(s):

Annet HAMMACHER ◽

John WIJDENES ◽

Douglas J. HILTON ◽

Nicos A. NICOLA ◽

Richard J. SIMPSON ◽

...

Keyword(s):

Proximal Region ◽

Leukaemia Inhibitory Factor ◽

Binding Affinities ◽

Wild Type ◽

Correct Orientation ◽

Fibronectin Type Iii ◽

Membrane Proximal Region ◽

Stimulating Factor ◽

Fibronectin Type

The receptor gp130 is used by the interleukin-6 (IL-6)-type cytokines, which include IL-6 and leukaemia-inhibitory factor (LIF). To investigate the role of the three extracellular membrane-proximal fibronectin-type-III-like (FNIII) modules of gp130 and the related receptor for granulocyte colony-stimulating factor (G-CSFR) in cytokine signal transduction we have transfected into murine myeloid M1-UR21 cells the chimaera (GR-FNIII)gp130, which contains the membrane-proximal FNIII modules of the G-CSFR on a gp130 backbone, and its complement, the chimaera (gp130-FNIII)GR. Whereas the binding affinities of 125I-labelled IL-6 to (GR-FNIII)gp130, or of 125I-Tyr1,3-G-CSF to (gp130-FNIII)GR, were similar to wild-type gp130 and wild-type G-CSFR, respectively, 125I-LIF failed to bind with high affinity to (GR-FNIII)gp130. In assays measuring differentiation the (gp130-FNIII)GR cells were fully responsive to G-CSF, whereas the (GR-FNIII)gp130 cells responded fully to the agonistic anti-gp130 monoclonal antibody (mAb) B-S12, but not to IL-6 or LIF. Neutralizing mAbs that recognize the membrane-proximal FNIII modules of gp130 or the G-CSFR differentially interfered with signalling by B-S12, LIF and G-CSF. The data suggest that B-S12 and G-CSF induce the correct orientation or conformation for signalling by the wild-type and chimaeric homodimeric receptors, that the membrane-proximal region of gp130 is important for the correct formation of the signalling IL-6-IL-6 receptor-gp130 complex and that this region is also involved in LIF-dependent receptor heterodimerization and signalling.

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Crosstalk between the Protein Surface and Hydrophobic Core in a Core-swapped Fibronectin Type III Domain

Journal of Molecular Biology ◽

10.1016/j.jmb.2007.10.056 ◽

2008 ◽

Vol 375 (2) ◽

pp. 560-571 ◽

Cited By ~ 17

Author(s):

Kate S. Billings ◽

Robert B. Best ◽

Trevor J. Rutherford ◽

Jane Clarke

Keyword(s):

Hydrophobic Core ◽

Protein Surface ◽

Type Iii ◽

Fibronectin Type Iii ◽

Fibronectin Type Iii Domain ◽

Fibronectin Type

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The Fibronectin Type-III (FNIII) Domain of ATF7IP Contributes to Efficient Transcriptional Silencing Mediated by The SETDB1 Complex

10.21203/rs.3.rs-44959/v1 ◽

2020 ◽

Author(s):

Takeshi Tsusaka ◽

Kei Fukuda ◽

Chikako Shimura ◽

Masaki Kato ◽

Yoichi Shinkai

Keyword(s):

Germ Cell ◽

Nuclear Localization ◽

Proteomic Analysis ◽

Embryonic Stem ◽

Transcriptional Silencing ◽

Binding Motif ◽

Type Iii ◽

Fibronectin Type Iii ◽

Fniii Domain ◽

Fibronectin Type

Abstract Background: The histone methyltransferase SETDB1 (also known as ESET) represses genes and various types of transposable elements, such as endogenous retroviruses (ERVs) and integrated exogenous retroviruses, through a deposition of trimethylation on lysine 9 of histone H3 (H3K9me3) in mouse embryonic stem cells (mESCs). ATF7IP (also known as MCAF1 or AM), a binding partner of SETDB1, regulates the nuclear localization and enzymatic activities of SETDB1 and plays a crucial role in SETDB1-mediated transcriptional silencing. In this study, we further dissected the ATF7IP function with its truncated mutants in Atf7ip knockout (KO) mESCs.Results: We demonstrated that the SETDB1-interaction region within ATF7IP is essential for ATF7IP-dependent SETDB1 nuclear localization and silencing of both ERVs and integrated retroviral transgenes, whereas its C-terminal fibronectin type-III (FNIII) domain is dispensable for both these functions; rather, it has a role in efficient silencing mediated by the SETDB1 complex. Proteomic analysis identified a number of FNIII domain-interacting proteins, some of which have a consensus binding motif. We showed that one of the FNIII domain-binding proteins, ZMYM2, was involved in the efficient silencing of a transgene by ATF7IP. RNA-seq analysis of Atf7ip KO and WT or the FNIII domain mutant of ATF7IP-rescued Atf7ip KO mESCs showed that the FNIII domain mutant re-silenced most de-repressed SETDB1/ATF7IP-targeted ERVs compared to the WT. However, the silencing activity of the FNIII domain mutant was weaker than that of the ATF7IP WT, and some of the de-repressed germ cell-related genes in Atf7ip KO mESCs were not silenced by the FNIII domain mutant. Such germ cell-related genes are targeted and silenced by the MAX/MGA complex, and MGA was also identified as another potential binding molecule of the ATF7IP FNIII domain in the proteomic analysis. This suggests that the FNIII domain of ATF7IP acts as a binding hub of ATF7IP-interacting molecules possessing a specific interacting motif we named FAM and contributes to one layer of the SETDB1/ATF7IP complex-mediated silencing mechanisms.Conclusions: Our findings contributed to further understanding the function of ATF7IP in the SETDB1 complex, revealed the role of the FNIII domain of ATF7IP in transcriptional silencing, and suggested a potential underlying molecular mechanism for it.

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Neurite Fasciculation Mediated by Complexes of Axonin-1 and Ng Cell Adhesion Molecule

The Journal of Cell Biology ◽

10.1083/jcb.143.6.1673 ◽

1998 ◽

Vol 143 (6) ◽

pp. 1673-1690 ◽

Cited By ~ 69

Author(s):

Stefan Kunz ◽

Marianne Spirig ◽

Claudia Ginsburg ◽

Andrea Buchstaller ◽

Philipp Berger ◽

...

Keyword(s):

Cell Adhesion ◽

Adhesion Molecule ◽

Extracellular Space ◽

Cell Adhesion Molecule ◽

Homophilic Binding ◽

Neural Cell Adhesion ◽

Fibronectin Type Iii ◽

Fniii Domain ◽

Cis Interaction ◽

Fibronectin Type

Neural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1–NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM–NgCAM interaction could be established simultaneously with the axonin-1–NgCAM interaction. In contrast, the axonin-1–NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.

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The fibronectin type-III (FNIII) domain of ATF7IP contributes to efficient transcriptional silencing mediated by the SETDB1 complex

Epigenetics & Chromatin ◽

10.1186/s13072-020-00374-4 ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Takeshi Tsusaka ◽

Kei Fukuda ◽

Chikako Shimura ◽

Masaki Kato ◽

Yoichi Shinkai

Keyword(s):

Germ Cell ◽

Nuclear Localization ◽

Proteomic Analysis ◽

Embryonic Stem ◽

Transcriptional Silencing ◽

Binding Motif ◽

Type Iii ◽

Fibronectin Type Iii ◽

Fniii Domain ◽

Fibronectin Type

Abstract Background The histone methyltransferase SETDB1 (also known as ESET) represses genes and various types of transposable elements, such as endogenous retroviruses (ERVs) and integrated exogenous retroviruses, through a deposition of trimethylation on lysine 9 of histone H3 (H3K9me3) in mouse embryonic stem cells (mESCs). ATF7IP (also known as MCAF1 or AM), a binding partner of SETDB1, regulates the nuclear localization and enzymatic activities of SETDB1 and plays a crucial role in SETDB1-mediated transcriptional silencing. In this study, we further dissected the ATF7IP function with its truncated mutants in Atf7ip knockout (KO) mESCs. Results We demonstrated that the SETDB1-interaction region within ATF7IP is essential for ATF7IP-dependent SETDB1 nuclear localization and silencing of both ERVs and integrated retroviral transgenes, whereas its C-terminal fibronectin type-III (FNIII) domain is dispensable for both these functions; rather, it has a role in efficient silencing mediated by the SETDB1 complex. Proteomic analysis identified a number of FNIII domain-interacting proteins, some of which have a consensus binding motif. We showed that one of the FNIII domain-binding proteins, ZMYM2, was involved in the efficient silencing of a transgene by ATF7IP. RNA-seq analysis of Atf7ip KO and WT or the FNIII domain mutant of ATF7IP-rescued Atf7ip KO mESCs showed that the FNIII domain mutant re-silenced most de-repressed SETDB1/ATF7IP-targeted ERVs compared to the WT. However, the silencing activity of the FNIII domain mutant was weaker than that of the ATF7IP WT, and some of the de-repressed germ cell-related genes in Atf7ip KO mESCs were not silenced by the FNIII domain mutant. Such germ cell-related genes are targeted and silenced by the MAX/MGA complex, and MGA was also identified as another potential binding molecule of the ATF7IP FNIII domain in the proteomic analysis. This suggests that the FNIII domain of ATF7IP acts as a binding hub of ATF7IP-interacting molecules possessing a specific interacting motif we named FAM and contributes to one layer of the SETDB1/ATF7IP complex-mediated silencing mechanisms. Conclusions Our findings contributed to further understanding the function of ATF7IP in the SETDB1 complex, revealed the role of the FNIII domain of ATF7IP in transcriptional silencing, and suggested a potential underlying molecular mechanism for it.

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Transient Opening of Fibronectin Type III (FNIII) Domains: The Interaction of the Third FNIII Domain of FN with Anastellin

Biochemistry ◽

10.1021/bi900001g ◽

2009 ◽

Vol 48 (19) ◽

pp. 4189-4197 ◽

Cited By ~ 15

Author(s):

Tomoo Ohashi ◽

Anne Marie Augustus ◽

Harold P. Erickson

Keyword(s):

Type Iii ◽

The Third ◽

Fibronectin Type Iii ◽

Fniii Domain ◽

Fibronectin Type

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Role of Binding of Plectin to the Integrin β4 Subunit in the Assembly of Hemidesmosomes

Molecular Biology of the Cell ◽

10.1091/mbc.e03-09-0697 ◽

2004 ◽

Vol 15 (3) ◽

pp. 1211-1223 ◽

Cited By ~ 84

Author(s):

J. Koster ◽

S. van Wilpe ◽

I. Kuikman ◽

S.H.M. Litjens ◽

A. Sonnenberg

Keyword(s):

Binding Sites ◽

Cytoplasmic Domain ◽

Actin Binding ◽

Actin Binding Domain ◽

Integrin Β4 ◽

Fibronectin Type Iii ◽

Intramolecular Association ◽

Two Hybrid ◽

Fibronectin Type

We have previously shown that plectin is recruited into hemidesmosomes through association of its actin-binding domain (ABD) with the first pair of fibronectin type III (FNIII) repeats and a small part of the connecting segment (residues 1328–1355) of the integrin β4 subunit. Here, we show that two proline residues (P1330 and P1333) in this region of the connecting segment are critical for supporting β4-mediated recruitment of plectin. Additional binding sites for the plakin domain of plectin on β4 were identified in biochemical and yeast two-hybrid assays. These sites are located at the end of the connecting segment (residues 1383–1436) and in the region containing the fourth FNIII repeat and the C-tail (residues 1570–1752). However, in cells, these additional binding sites cannot induce the assembly of hemidesmosomes without the interaction of the plectin-ABD with β4. Because the additional plectin binding sites overlap with sequences that mediate an intramolecular association of the β4 cytoplasmic domain, we propose that they are not accessible for binding and need to become exposed as the result of the binding of the plectin-ABD to β4. Furthermore, these additional binding sites might be necessary to position the β4 cytoplasmic domain for an optimal interaction with other hemidesmosomal components, thereby increasing the efficiency of hemidesmosome assembly.

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The fibronectin type-III (FNIII) domain of ATF7IP contributes to efficient transcriptional silencing mediated by the SETDB1 complex

10.21203/rs.3.rs-44959/v2 ◽

2020 ◽

Author(s):

Yoichi Shinkai ◽

Takeshi Tsusaka ◽

Kei Fukuda ◽

Chikako Shimura ◽

Masaki Kato

Keyword(s):

Germ Cell ◽

Nuclear Localization ◽

Proteomic Analysis ◽

Embryonic Stem ◽

Transcriptional Silencing ◽

Binding Motif ◽

Type Iii ◽

Fibronectin Type Iii ◽

Fniii Domain ◽

Fibronectin Type

Abstract Background: The histone methyltransferase SETDB1 (also known as ESET) represses genes and various types of transposable elements, such as endogenous retroviruses (ERVs) and integrated exogenous retroviruses, through a deposition of trimethylation on lysine 9 of histone H3 (H3K9me3) in mouse embryonic stem cells (mESCs). ATF7IP (also known as MCAF1 or AM), a binding partner of SETDB1, regulates the nuclear localization and enzymatic activities of SETDB1 and plays a crucial role in SETDB1-mediated transcriptional silencing. In this study, we further dissected the ATF7IP function with its truncated mutants in Atf7ip knockout (KO) mESCs.Results: We demonstrated that the SETDB1-interaction region within ATF7IP is essential for ATF7IP-dependent SETDB1 nuclear localization and silencing of both ERVs and integrated retroviral transgenes, whereas its C-terminal fibronectin type-III (FNIII) domain is dispensable for both these functions; rather, it has a role in efficient silencing mediated by the SETDB1 complex. Proteomic analysis identified a number of FNIII domain-interacting proteins, some of which have a consensus binding motif. We showed that one of the FNIII domain-binding proteins, ZMYM2, was involved in the efficient silencing of a transgene by ATF7IP. RNA-seq analysis of Atf7ip KO and WT or the FNIII domain mutant of ATF7IP-rescued Atf7ip KO mESCs showed that the FNIII domain mutant re-silenced most de-repressed SETDB1/ATF7IP-targeted ERVs compared to the WT. However, the silencing activity of the FNIII domain mutant was weaker than that of the ATF7IP WT, and some of the de-repressed germ cell-related genes in Atf7ip KO mESCs were not silenced by the FNIII domain mutant. Such germ cell-related genes are targeted and silenced by the MAX/MGA complex, and MGA was also identified as another potential binding molecule of the ATF7IP FNIII domain in the proteomic analysis. This suggests that the FNIII domain of ATF7IP acts as a binding hub of ATF7IP-interacting molecules possessing a specific interacting motif we named FAM and contributes to one layer of the SETDB1/ATF7IP complex-mediated silencing mechanisms.Conclusions: Our findings contributed to further understanding the function of ATF7IP in the SETDB1 complex, revealed the role of the FNIII domain of ATF7IP in transcriptional silencing, and suggested a potential underlying molecular mechanism for it.

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X-ray structures of Nfs2, the plastidial cysteine desulfurase fromArabidopsis thaliana

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14017026 ◽

2014 ◽

Vol 70 (9) ◽

pp. 1180-1185 ◽

Cited By ~ 8

Author(s):

Thomas Roret ◽

Henri Pégeot ◽

Jérémy Couturier ◽

Guillermo Mulliert ◽

Nicolas Rouhier ◽

...

Keyword(s):

Binding Sites ◽

Wild Type ◽

Cysteine Desulfurase ◽

Iron Sulfur Cluster ◽

X Ray ◽

Iron Sulfur ◽

Group I ◽

Catalytic Cysteine ◽

Group Ii ◽

Wild Type Form

The chloroplasticArabidopsis thalianaNfs2 (AtNfs2) is a group II pyridoxal 5′-phosphate-dependent cysteine desulfurase that is involved in the initial steps of iron–sulfur cluster biogenesis. The group II cysteine desulfurases require the presence of sulfurtransferases such as SufE proteins for optimal activity. Compared with group I cysteine desulfurases, proteins of this group contains a smaller extended lobe harbouring the catalytic cysteine and have a β-hairpin constraining the active site. Here, two crystal structures of AtNfs2 are reported: a wild-type form with the catalytic cysteine in a persulfide-intermediate state and a C384S variant mimicking the resting state of the enzyme. In both structures the well conserved Lys241 covalently binds pyridoxal 5′-phosphate, forming an internal aldimine. Based on available homologous bacterial complexes, a model of a complex between AtNfs2 and the SufE domain of its biological partner AtSufE1 is proposed, revealing the nature of the binding sites.

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