Mouse Model of Fragile X Syndrome Analyzed by Quantitative Proteomics: A Comparison of Methods.

Multiple methods for quantitative proteomics are available for proteome profiling. It is unclear which methods are most useful in situations involving deep proteome profiling and the detection of subtle distortions in the proteome. Here, we compared the performance of seven different strategies in the analysis of a mouse model of Fragile X Syndrome, involving the knockout of the fmr1 gene that is the leading cause of autism spectrum disorder. Focusing on the cerebellum, we show that data-independent acquisition (DIA) and the tandem mass tag (TMT)-based real-time search method (RTS) generated the most informative profiles, generating 334 and 329 significantly altered proteins, respectively, although the latter still suffered from ratio compression. Label-free methods such as BoxCar and a conventional data-dependent acquisition were too noisy to generate a reliable profile, while TMT methods that do not invoke RTS showed a suppressed dynamic range. The TMT method using the TMTpro reagents together with complementary ion quantification (ProC) overcomes ratio compression, but current limitations in ion detection reduce sensitivity. Overall, both DIA and RTS uncovered known regulators of the syndrome and detected alterations in calcium signaling pathways that are consistent with calcium deregulation recently observed in imaging studies. Data are available via ProteomeXchange with the identifier PXD039885.

Motif orientation matters: Structural characterization of TEAD1 recognition of genomic DNA.

TEAD transcription factors regulate gene expression through interactions with DNA and other proteins. They are crucial for the development of eukaryotic organisms and to control the expression of genes involved mostly in cell proliferation and differentiation; however, their deregulation can lead to tumorigenesis. To study the interactions of TEAD1 with M-CAT motifs and their inverted versions, the KD of each complex was determined, and H/D exchange, quantitative chemical cross-linking, molecular docking, and smFRET were utilized for structural characterization. ChIP-qPCR was employed to correlate the results with a cell line model. The results obtained showed that although the inverted motif has 10× higher KD, the same residues were affected by the presence of M-CAT in both orientations. Molecular docking and smFRET revealed that TEAD1 binds the inverted motif rotated 180°. In addition, the inverted motif was proven to be occupied by TEAD1 in Jurkat cells, suggesting that the low-affinity binding sites present in the human genome may possess biological relevance.

Studying Protein-DNA Interactions by Hydrogen/Deuterium Exchange Mass Spectrometry.

Protein hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) can be used to study interactions of proteins with various ligands, to describe the effects of mutations, or to reveal structural responses of proteins to different experimental conditions. It is often described as a method with virtually no limitations in terms of protein size or sample composition. While this is generally true, there are, however, ligands or buffer components that can significantly complicate the analysis. One such compound, that can make HDX-MS troublesome, is DNA. In this chapter, we will focus on the analysis of protein-DNA interactions, describe the detailed protocol, and point out ways to overcome the complications arising from the presence of DNA.

MS-Based Approaches Enable the Structural Characterization of Transcription Factor/DNA Response Element Complex.

The limited information available on the structure of complexes involving transcription factors and cognate DNA response elements represents a major obstacle in the quest to understand their mechanism of action at the molecular level. We implemented a concerted structural proteomics approach, which combined hydrogen-deuterium exchange (HDX), quantitative protein-protein and protein-nucleic acid cross-linking (XL), and homology analysis, to model the structure of the complex between the full-length DNA binding domain (DBD) of Forkhead box protein O4 (FOXO4) and its DNA binding element (DBE). The results confirmed that FOXO4-DBD assumes the characteristic forkhead topology shared by these types of transcription factors, but its binding mode differs significantly from those of other members of the family. The results showed that the binding interaction stabilized regions that were rather flexible and disordered in the unbound form. Surprisingly, the conformational effects were not limited only to the interface between bound components, but extended also to distal regions that may be essential to recruiting additional factors to the transcription machinery. In addition to providing valuable new insights into the binding mechanism, this project provided an excellent evaluation of the merits of structural proteomics approaches in the investigation of systems that are not directly amenable to traditional high-resolution techniques.