Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structural basis of human full-length kindlin-3 homotrimer in an auto-inhibited state

ABSTRACT: Kindlin-1, -2, and -3, directly bind integrin β cytoplasmic tails to regulate integrin activation and signaling. Despite their functional significance and link to several diseases, structural information on full-length kindlin proteins remains unknown. Here, we report the crystal structure of human full-length kindlin-3, which reveals a novel homotrimer state. Unlike kindlin-3 monomer, which is the major population in insect and mammalian cell expression systems, kindlin-3 trimer does not bind integrin β cytoplasmic tail as the integrin-binding pocket in the F3 sub-domain of one protomer is occluded by the PH domain of another protomer, suggesting that kindlin-3 is auto-inhibited upon trimer formation. This is also supported by functional assays in which kindlin-3 knockout K562 erythroleukemia cells re-constituted with the mutant kindlin-3 containing trimer-disrupting mutations exhibited an increase in integrin-mediated adhesion and spreading on fibronectin compared to those re-constituted with wild type kindlin-3. Taken together, our findings reveal a novel mechanism of kindlin auto-inhibition that involves its homotrimer formation.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: SoFong Cam Ngan

LAB HEAD: Siu Kwan Sze

PROVIDER: PXD019110 | Pride | 2020-06-08

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	Annotated_XL_MSMS_Spectra_AGDALWLRFKYYSFFDLDPK10-LASKGR4.pdf	Pdf
	Annotated_XL_MSMS_Spectra_IDLAVGDVVKTWR10-KDEILGIANNR1.pdf	Pdf
	Annotated_XL_MSMS_Spectra_KDEILGIANNR1-LASKGR4.pdf	Pdf
	Annotated_XL_MSMS_Spectra_YYSFFDLDPKTDPVR10-LASKGR4.pdf	Pdf
	Individual_Annotated_XL_MSMS_Spectra.dat	Other

Items per page:

1 - 5 of 8

Similar Datasets

Expression data of cDNA from the ACHN control and Kindlin-2-depleting cells

Project description:Kindlin-2, an integrin-interacting FERM-domain-containing protein, has been known to play critical roles for tumor progression. However, the role of Kindlin-2 in renal cell carcinoma (RCC) progression has not been reported. We aim to investigate the role of Kindlin-2 in the progression of RCC and the underlying mechanisms. To uncover the related pathway in which Kindlin-2 is involved to promote clear cell renal cell carcinoma progression, ACHN control and Kindlin-2-depleting cells were analyzed by Affymetrix GeneChip human Gene 1.0 ST Arrays. ACHN cells were transfected with control short hairpin RNA (shRNA) or Kindlin-2 shRNA. ACHN control and Kindlin-2-depleting cells cDNAs were hybridized to Affymetrix GeneChip Human Gene 1.0 ST arrays. Data were analyzed by Expression Console 1.4.1.

2015-12-16 | E-GEOD-76020 | biostudies-arrayexpress

Expression data of cDNA from the ACHN control and Kindlin-2-depleting cells

2015-12-16 | GSE76020 | GEO

Integrin b1 acetylation-induced transcriptomic reprograming of mouse embryonic endothelial cells and functions

Project description:Reciprocal regulation of integrin-dependent cell-matrix adhesions and cell-cell junctions is critical for controlling endothelial permeability and proliferation in cancer and inflammatory diseases, but remain poorly understood. Here, we investigated how acetylation of the distal NPKY-motif of b1-integrin influences endothelial cell physiology and barrier function. Expression of an acetylation-mimetic B1A-K794Q-GFP mutant induced transcriptomic reprograming of embryonic endothelial cells particularly in genes responsible for cell adhesion, proliferation, polarity and barrier function. B1A-K794Q-GFP induced a constitutive activation of MAPK signaling, an impairment of junctions, increased proliferation and an altered contact inhibition at confluency. Structural analysis of integrin b1 interaction with KINDLIN-2, biochemical pulldown assay and molecular dynamic analysis showed that acetylation of K794 increases KINDLIN-2 binding to the integrin b1 cytoplasmic tail. Accordingly, altering KINDLIN-2 levels modified Claudin-5 expression and endothelial barrier function. Revealing how integrin b1 acetylation regulates endothelial cell junctions offers new therapeutic possibilities for controlling vascular permeability.

2024-01-01 | GSE238019 | GEO

Low kindlin-3 levels in osteoclasts of kindlin-3 hypomorphic mice result in osteopetrosis due to leaky sealing zones

Project description:Osteoclasts form special integrin-mediated adhesion structures called sealing zones that enable them to adhere to and resorb bone. Sealing zones consist mainly of densely packed podosomes tightly inter-connected by actin fibers. Their formation requires the presence of the hematopoietic integrin regulator kindlin-3. In the current study, we investigated osteoclasts and their adhesion structures in kindlin-3 hypomorphic mice expressing only 5-10% of kindlin-3. Low kindlin-3 expression reduces integrin activity, results in impaired osteoclast adhesion and signaling, and delays cell spreading. Despite these defects, in vitro generated kindlin-3-hypomorphic osteoclasts arrange their podosomes into adhesion patches and belts, which show abnormal podosome and actin organization. Remarkably, kindlin-3-hypomorphic osteoclasts form sealing zones when cultured on calcified matrix in vitro and on bone surface in vivo. However, functional assays as well as immunohistochemical staining and electron micrographs of bone sections showed that they fail to properly seal the resorption lacunae, which is required for secreted proteases to be able to digest bone matrix. This results in mild osteopetrosis. Our study reveals a new, hitherto understudied function of kindlin-3 as an essential organizer of integrin-mediated adhesion structures, such as sealing zones.

2021-11-25 | PXD026711 | Pride

Transcriptional Analysis of the Effects of Integrin-driven Axon Regeneration in the Spinal Cord

Project description:To elucidate the mechanisms and down-stream pathways affected by activated integrin expression, we performed transcriptomic analyses of dorsal root gangliona (DRG) sensory neurons transduced with α9 integrin and its activator kindlin-1(α9k1), which enable axons to interact with tenascin-C, or controls, with and without dorsal root/central axotomy.

2022-11-30 | GSE188775 | GEO

Activation of automethylated PRC2 by dimerization on chromatin

Project description:Polycomb Repressive Complex 2 (PRC2) trimethylates lysine 27 of histone 3 (H3K27me3). This modification transcriptionally represses chromatin and its dynamics are essential for embryonic development. PRC2 also methylates its cofactor JARID2 and both methylated H3 tail and JARID2 allosterically activate the complex. PRC2 undergoes auto-methylation of its catalytic subunit, which stimulates its activity by an unknown mechanism. We show that automethylated PRC2 forms a dimer on substrate chromatin that results in autoactivation in trans. An inactive PRC2 protomer distal to the substrate nucleosome serves as an allosteric activator of the second PRC2 protomer, which is poised to methylate H3. Functional assays support our model of allosteric activation via dimerization, suggesting a novel mechanism of PRC2 trans-autoactivation that may be important for installation of H3K27me3 into naïve chromatin.

2023-11-13 | GSE234793 | GEO

The integrin linked kinase is required for chemokine-triggered high affinity conformation of the leukocyte β2-integrin LFA-1The integrin linked kinase is required for chemokine-triggered high affinity conformation of the leukocyte β2-integrin LFA-1

Project description:Invasion of leukocytes, including neutrophils, in response to injury or infection relies on the orchestrated activation of integrins. The neutrophil integrin lymphocyte function-associated antigen-1 (LFA-1) has been implicated in the regulation of leukocyte adhesion by binding to ICAM-1 expressed on activated endothelial cells. The activation-dependent conformational change of LFA-1 to the high affinity conformation (H+) requires kindlin-3 binding to the tail of the β2-integrin. How the integrin linked kinase (ILK) affects activation of β2-integrins in leukocytes is currently unknown. Here, utilizing in vitro microfluidic adhesion chambers with conformation specific antibodies for neutrophil-like HL-60 cells, we show that knockdown of ILK reduces the conformational change of β2-integrins to the H+ conformation. Consequently, ILK-deficient mice show defects of leukocyte adhesion and recruitment in a chemokine and integrin-dependent cremaster muscle model and in a clinically relevant model of renal-ischemia-reperfusion-injury. Absence of protein kinase C (PKC)-α, which is known to phosphorylate Kindlin-3, reproduces such phenotype in bone marrow chimeric mice. ILK is required for chemokine-induced upregulation of PKC-α activity. Mass spectrometry analysis and western blot analyses revealed a stimulation- and ILK-dependent phosphorylation of kindlin-3 upon activation. Our data thus show that ILK impacts kindlin-3-dependent conformational activation of LFA-1, thus contributing to an inflammatory response.

2020-07-20 | PXD017088 | Pride

Rational correction of pathogenic conformational defects in HTRA1

Project description:Loss-of-function mutations in the homotrimeric serine protease HTRA1 cause cerebral vasculopathy. Here, we show that disease-causing mutations targeting the protomer-protomer interface impair trimerization. Focusing on a prototypical interface mutation (R274Q), we designed an HTRA1 variant that complemented pathogenic HTRA1 and reconstituted its multimeric assembly and enzymatic activity in vitro. Genetic experiments in Htra1R274Q mice further demonstrated that expression of this protein-based corrector in trans was sufficient to stabilize HTRA1-R274Q and restore the cerebrovascular proteome. As an alternative approach to achieve repair of pathogenic interface mutants, we generated supramolecular chemical ligands that shifted the monomer-trimer equilibrium by stabilizing proteolytically active trimers. Moreover, we identified a peptidic ligand that activated HTRA1 monomers. Collectively, our findings open novel perspectives for tailored protein repair strategies.

2023-11-24 | PXD024683 | Pride

Dynamics of different mGBP as revealed by computational and experimental methods

Project description:Guanylate binding proteins (GBP) belong to the superfamily of dynamin proteins. Those might assemble into larger structures upon initial dimerization. We analyzed monomeric and dimerization of mGBP7 via molecular dynamic simulations and compared the developed models with data from crosslinking mass spectrometry experiments.

2024-01-26 | PXD026979 | Pride

Integrative Threading of the DNA-PKcs Disordered Region with Crosslinking Restraints

Project description:Integrative structure modelling was applied to understand how auto-phosphorylation of the ABCDE cluster in DNA-PKcs can occur. We used crosslinking restraints to assign sequence to structurally unassigned helices in DNA-PKcs s in the largely disordered region containing the ABCDE cluster. Our final model positions the ABCDE cluster at the end of the second helices, suggesting that phosphorylation occurs through trans-auto-phosphorylation, or through a large conformational change in the disordered region for cis-auto-phosphorylation.

2019-12-10 | PXD016639 | Pride