NDLI: Identification of an FHL1 protein complex containing ACTN1, ACTN4, and PDLIM1 using affinity purifications and MS-based protein-protein interaction analysis.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Sharma, Parveen Shathasivam, Thiruchelvi Ignatchenko, Vladimir Kislinger, Thomas Gramolini, Anthony O.
Description	Country affiliation: Canada Author Affiliation: Sharma P ( Department of Physiology, University of Toronto, Toronto, Ontario, Canada.)
Abstract	INTRODUCTION: Four and a half LIM domains protein 1 (FHL1) is the most widely expressed member of the FHL family of proteins, consisting of four and a half highly conserved LIM domains. A multifunctional and integral role for FHL1 has been implicated in muscle development, structural maintenance, and signaling. To date, 27 FHL1 mutations have been identified that result in at least six different X-linked myopathies, with patients often presenting with cardiovascular complications. Since proteins assemble into dynamic complexes within the cell, FHL1 likely mediates its biological functions in conjunction with other proteins. Delineation of FHL1 interactions could provide insight into its regulatory functions. METHODS: We performed tandem affinity purification from human embryonic kidney 293 (HEK-293) cells to purify FHL1 and interacting proteins. To identify the potential interactors of FHL1 we performed a total of 9 different purifications from HEK-293 cells which included 3 experimental replicates for each biological condition: FHL1, tag control (DPYSL3), and negative control (empty vector). Purified samples were analyzed by liquid chromatography mass spectrometry (LC-MS). Potential interactors were then verified by immunoprecipitation from mouse heart ventricles and interactions visualized in adult cardiomyocytes using 3D fluorescence microscopy. RESULTS: We identified a total of 310 different proteins from all 9 purifications and by applying stringent filtering criteria we eliminated all proteins found in any of the controls and only allowed those that were detected in two or more bait purification. We identified 34 high confidence potential binding partners of FHL1. We then showed that FHL1 exists as part of a complex that binds with PDLIM1, GSN and ACTN1.
File Format	HTM / HTML
ISSN	1742206X
e-ISSN	17422051
DOI	10.1039/c0mb00235f
Journal	Molecular BioSystems
Issue Number	4
Volume Number	7
Language	English
Publisher	Royal Society of Chemistry
Publisher Date	2011-04-01
Publisher Place	Great Britain (UK)
Access Restriction	Open
Subject Keyword	Discipline Molecular Biology Discipline Biochemistry Actinin Chromatography, Affinity Intracellular Signaling Peptides And Proteins Mass Spectrometry Microfilament Proteins Muscle Proteins Metabolism Animals Computational Biology Gelsolin Hek293 Cells Isolation & Purification Lim Domain Proteins Mice Protein Binding Recombinant Fusion Proteins Transcription Factors Research Support, Non-u.s. Gov't
Content Type	Text
Resource Type	Article
Subject	Molecular Biology Biotechnology

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in