Tandem affinity purification of histones, coupled to mass spectrometry, identifies associated proteins and new sites of post-translational modification in Saccharomyces cerevisiae

2016 ◽  
Vol 136 ◽  
pp. 183-192 ◽  
Author(s):  
M. Luz Valero ◽  
Ramon Sendra ◽  
Mercè Pamblanco
Keyword(s):  
Mass Spectrometry ◽  
Saccharomyces Cerevisiae ◽  
Affinity Purification ◽  
Tandem Affinity Purification ◽  
Post Translational Modification ◽  
Associated Proteins

scholarly journals Tandem Affinity Purification and Mass Spectrometry (TAP‐MS) for the Analysis of Protein Complexes

2019 ◽  
Vol 96 (1) ◽  
Author(s):  
Guillaume Adelmant ◽  
Brijesh K. Garg ◽  
Maria Tavares ◽  
Joseph D. Card ◽  
Jarrod A. Marto
Keyword(s):  
Mass Spectrometry ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Tandem Affinity Purification

scholarly journals Post-translational modification of nucleoid-associated proteins: an extra layer of functional modulation in bacteria?

2018 ◽  
Vol 46 (5) ◽  
pp. 1381-1392 ◽  
Author(s):  
Ivar W. Dilweg ◽  
Remus T. Dame
Keyword(s):  
Mass Spectrometry ◽  
Genetic Information ◽  
Structural Integrity ◽  
Mass Spectrometry Data ◽  
Post Translational Modification ◽  
Functional Importance ◽  
Widespread Occurrence ◽  
Associated Proteins ◽  
Chromatin Folding ◽  
Functional Modulation

Post-translational modification (PTM) of histones has been investigated in eukaryotes for years, revealing its widespread occurrence and functional importance. Many PTMs affect chromatin folding and gene activity. Only recently the occurrence of such modifications has been recognized in bacteria. However, it is unclear whether PTM of the bacterial counterparts of eukaryotic histones, nucleoid-associated proteins (NAPs), bears a comparable significance. Here, we scrutinize proteome mass spectrometry data for PTMs of the four most abundantly present NAPs in Escherichia coli (H-NS, HU, IHF and FIS). This approach allowed us to identify a total of 101 unique PTMs in the 11 independent proteomic studies covered in this review. Combined with structural and genetic information on these proteins, we describe potential effects of these modifications (perturbed DNA-binding, structural integrity or interaction with other proteins) on their function.

scholarly journals Mono- and Biallelic Protein-Truncating Variants in Alpha-Actinin 2 Cause Cardiomyopathy Through Distinct Mechanisms

2021 ◽  
Author(s):  
Malene E. Lindholm ◽  
David Jimenez-Morales ◽  
Han Zhu ◽  
Kinya Seo ◽  
David Amar ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Structural Changes ◽  
Affinity Purification ◽  
Protein Protein Interaction ◽  
Cellular Hypertrophy ◽  
C Terminus ◽  
Associated Proteins ◽  
Cardiac Sarcomeres ◽  
Affinity Purification Mass Spectrometry

Background: ACTN2 (alpha-actinin 2) anchors actin within cardiac sarcomeres. The mechanisms linking ACTN2 mutations to myocardial disease phenotypes are unknown. Here, we characterize patients with novel ACTN2 mutations to reveal insights into the physiological function of ACTN2. Methods: Patients harboring ACTN2 protein-truncating variants were identified using a custom mutation pipeline. In patient-derived iPSC-cardiomyocytes, we investigated transcriptional profiles using RNA sequencing, contractile properties using video-based edge detection, and cellular hypertrophy using immunohistochemistry. Structural changes were analyzed through electron microscopy. For mechanistic studies, we used coimmunoprecipitation for ACTN2, followed by mass-spectrometry to investigate protein-protein interaction, and protein tagging followed by confocal microscopy to investigate introduction of truncated ACTN2 into the sarcomeres. Results: Patient-derived iPSC-cardiomyocytes were hypertrophic, displayed sarcomeric structural disarray, impaired contractility, and aberrant Ca 2+ -signaling. In heterozygous indel cells, the truncated protein incorporates into cardiac sarcomeres, leading to aberrant Z-disc ultrastructure. In homozygous stop-gain cells, affinity-purification mass-spectrometry reveals an intricate ACTN2 interactome with sarcomere and sarcolemma-associated proteins. Loss of the C-terminus of ACTN2 disrupts interaction with ACTN1 and GJA1, 2 sarcolemma-associated proteins, which may contribute to the clinical arrhythmic and relaxation defects. The causality of the stop-gain mutation was verified using CRISPR-Cas9 gene editing. Conclusions: Together, these data advance our understanding of the role of ACTN2 in the human heart and establish recessive inheritance of ACTN2 truncation as causative of disease.

scholarly journals The Aryl-Hydrocarbon Receptor Protein Interaction Network (AHR-PIN) as Identified by Tandem Affinity Purification (TAP) and Mass Spectrometry

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Dorothy M. Tappenden ◽  
Hye Jin Hwang ◽  
Longlong Yang ◽  
Russell S. Thomas ◽  
John J. LaPres
Keyword(s):  
Mass Spectrometry ◽  
Gene Regulation ◽  
Aryl Hydrocarbon Receptor ◽  
Protein Interaction ◽  
Protein Interaction Network ◽  
Affinity Purification ◽  
Interaction Network ◽  
Tandem Affinity Purification ◽  
Toxic Response ◽  
Aryl Hydrocarbon

The aryl-hydrocarbon receptor (AHR), a ligand activated PAS superfamily transcription factor, mediates most, if not all, of the toxicity induced upon exposure to various dioxins, dibenzofurans, and planar polyhalogenated biphenyls. While AHR-mediated gene regulation plays a central role in the toxic response to dioxin exposure, a comprehensive understanding of AHR biology remains elusive. AHR-mediated signaling starts in the cytoplasm, where the receptor can be found in a complex with the heat shock protein of 90 kDa (Hsp90) and the immunophilin-like protein, aryl-hydrocarbon receptor-interacting protein (AIP). The role these chaperones and other putative interactors of the AHR play in the toxic response is not known. To more comprehensively define the AHR-protein interaction network (AHR-PIN) and identify other potential pathways involved in the toxic response, a proteomic approach was undertaken. Using tandem affinity purification (TAP) and mass spectrometry we have identified several novel protein interactions with the AHR. These interactions physically link the AHR to proteins involved in the immune and cellular stress responses, gene regulation not mediated directly via the traditional AHR:ARNT heterodimer, and mitochondrial function. This new insight into the AHR signaling network identifies possible secondary signaling pathways involved in xenobiotic-induced toxicity.

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