Biology subjects

Sudhamalla, B.

Publications and source records attributed to Sudhamalla, B..

6 recordsLinked to original sources

bioRxiv2026

Mapping the Nonhistone Interactome of the TRIM24 Bromodomain Using Genetically Encoded Complementary Photocrosslinkers

TRIM24 is a tandem epigenetic reader containing plant homeodomain (PHD) and bromodomain modules in its C-terminal region. The PHD domain recognizes unmodified lysine 4 of histone H3, whereas the bromodomain recognizes acetylated histones H3 and H4. However, whether the TRIM24 bromodomain also engages acetylated nonhistone proteins remains poorly understood. To address this question, we developed site-specific photocrosslinking probes for the TRIM24 bromodomain using interaction-based protein profiling (IBPP). 4-azido-L-phenylalanine (AzF) and 4-benzoyl-L-phenylalanine (BzF) were genetically incorporated at L922, a residue positioned within the acetyllysine-binding pocket. Both variants preserved recognition of acetylated histone peptides and underwent efficient UV-dependent photocrosslinking. Quantitative proteomics of photocaptured proteins revealed distinct profiles for the two probes, identifying NPM1 among the L922AzF-enriched proteins and CDK9 among the L922BzF-enriched proteins. Additional candidate proteins included MAP4, RAN, and SND1. ChIP-seq analysis showed overlapping genomic enrichment of TRIM24, CDK9, and NPM1 near transcription start sites of cancer-associated genes. KEGG analysis further placed the identified proteins in several signalling pathways. Collectively, this study demonstrates that site-specific photocrosslinking can expand interactome analysis of epigenetic reader domains and provides candidate nonhistone proteins associated with the TRIM24 bromodomain for further investigation.

biochemistry↗

bioRxiv2025

Aromatic cage-directed azide-methyllysine photochemistry for profiling non-histone interacting partners of the MeCP2 methyl-CpG binding domain

Methyl-CpG binding protein 2 (MeCP2) is a well-characterized DNA methylation reader that has recently been shown to interact with methylated histones. MeCP2 recognizes tri-methylated lysine 27 on histone H3 (H3K27me3) through an aromatic cage within its methyl-CpG binding domain (MBD), and this interaction contributes to the regulation of MeCP2 target genes. However, whether MeCP2 can bind methylated non-histone proteins remains unknown. In this study, we sought to identify novel, aromatic cage-dependent non-histone interacting partners of MeCP2 using an unnatural amino acid-based photocrosslinking chemoproteomic strategy. We engineered the aromatic cage of MeCP2-MBD by incorporating the photocrosslinkable amino acid 4-azido-L-phenylalanine (AzF). The AzF-incorporated MeCP2-MBD variants efficiently crosslinked with histone H3 and with methylated histone marks such as H3K4me3 and H3K27me3. MeCP2-MBD-F142AzF was subsequently used to crosslink proteins from HEK293T cell lysates, and the enriched complexes were analyzed by mass spectrometry. This approach identified numerous previously unrecognized interacting partners of MeCP2, many of which are involved in key cellular processes including chromatin regulation, RNA processing, translation, and metabolism. These findings reveal that MeCP2 functions extend beyond DNA methylation reading and transcriptional repression, highlighting a broader role for MeCP2 in coordinating cellular homeostasis.

biochemistry↗

bioRxiv2025

Molecular Insights into Methyllysine Recognition by the Methyl-CpG-Binding Domain of MeCP2

Methyl-CpG binding protein 2 (MeCP2) is a DNA methylation reader, which is highly expressed in the central nervous system. Loss of function mutation of MeCP2, which impairs DNA binding, causes Rett syndrome, while gain of MeCP2 function causes MeCP2 duplication syndrome. MeCP2 functions as both transcription activator and repressor but its full range of activity remains largely unknown. Recent study suggests that beyond DNA binding, MBD domain of MeCP2 also interacts with methylated lysine residues on histone H3 tail. In this study we aimed to characterize the methyllysine binding pocket of MeCP2-MBD. Structural analysis reveals the presence of an aromatic cage formed by five residues, namely, W104, F132, Y141, F142, and F155, where the trimethyllysine of H3K27me3 binds, as confirmed in docking studies. Alanine substitution of these residues abolished binding of MeCP2-MBD with its reported ligand H3K27me3 in ITC and pull-down experiments. Genomic analysis of publicly available ChIP-seq data reveals that MeCP2 localizes with canonical histone methylation marks like H3K4me3, H3K9me3, H3K27me3 and H4K20me3 and regulate critical biological and cancer related pathways. Promoters of MeCP2 regulated genes, show signature of H3K4me3 and H3K27me3. In the genomic level, mutation of aromatic cage residue W104A, significantly reduced chromatin occupancy of MeCP2 as demonstrated by ChIP-qPCR experiments. Furthermore, our qRT-PCR experiment, shows that this aromatic cage dependent binding is necessary for regulating expression of MeCP2 target genes. Overall, our study establishes MeCP2 as a histone methylation reader and highlights the functional significance of its histone binding capacity in chromatin association and gene regulation.

biochemistry↗

bioRxiv2022

Identification of novel natural product inhibitors of BRD4 using high throughput virtual screening and MD simulation

Bromodomains are evolutionarily conserved structural motifs that recognize acetylated lysine residues on histone tails. They play a crucial role in shaping chromatin architecture and regulating gene expression in various biological processes. Mutations in bromodomains containing proteins leads to multiple human diseases, which makes them attractive target for therapeutic intervention. Extensive studies have been done on BRD4 as a target for several cancers, such as Acute Myeloid Leukemia (AML) and Burkitt Lymphoma. Several potential inhibitors have been identified against the BRD4 bromodomain. However, most of these inhibitors have drawbacks such as nonspecificity and toxicity, decreasing their appeal and necessitating the search for novel non-toxic inhibitors. This study aims to address this need by virtually screening natural compounds from the NPASS database against the Kac binding site of BRD4-BD1 using high throughput molecular docking followed by similarity clustering, pharmacokinetic screening, MD simulation, and MM-PBSA binding free energy calculations. Using this approach, we identified five natural product inhibitors having a similar or better binding affinity to the BRD4 bromodomain compared to JQ1 (previously reported inhibitor of BRD4). Further systematic analysis of these inhibitors resulted in the top three hits: NPC268484 (Palodesangren-B), NPC295021 (Candidine), and NPC313112 (Buxifoliadine-D). Collectively, our in silico results identified some promising natural products that have the potential to act as potent BRD4-BD1 inhibitors and can be considered for further validation through future in vitro and in vivo studies.

bioinformatics↗

bioRxiv2022

Molecular insights into the recognition of acetylated histone modifications by the BRPF2 bromodomain

HBO1 (HAT bound to ORC), a member of the MYST family of histone acetyltransferases (HATs), was initially identified as a binding partner of the origin recognition complex (ORC) that acetylates free histone H3, H4, and nucleosomal H3. It functions as a quaternary complex with the BRPF (BRPF1/2/3) scaffolding protein and two accessory proteins, ING4/5 and Eaf6. BRPF2 interaction with HBO1 has been shown to be important for regulating H3K14 acetylation during embryonic development. However, how the BRPF2 directs the HBO1 HAT complex to chromatin to regulate its HAT activity towards nucleosomal substrates remains unclear. Our findings reveal novel interacting partners of the BRPF2 bromodomain that recognizes different acetyllysine residues on the N-terminus of histone H4, H3, and H2A and preferentially binds to H4K5ac, H4K8ac, and H4K5acK12ac modifications. Further, mutational analysis of BRPF2 bromodomain coupled with ITC binding and pull-down assays on the histone substrates identified critical residues responsible for acetyllysine binding. Moreover, the BRPF2 bromodomain could enrich H4K5ac mark-bearing mononucleosomes compared to other acetylated H4 marks. Consistent with this, ChIP-seq analysis revealed that BRPF2 strongly co-localizes with HBO1 at histone H4K5ac and H4K8ac marks near the TSS in the genome. Together, our study provides novel insights into how the histone binding function of the BRPF2 bromodomain directs the recruitment of the HBO1 HAT complex to chromatin to regulate gene expression.

biochemistry↗

bioRxiv2021

Uncovering the Bromodomain Interactome using Site-Specific Azide-Acetyllysine Photochemistry, Proteomic Profiling and Structural Characterization

Protein-protein interactions mediated by acetyllysines and bromodomains are essential for the regulation of eukaryotic gene expression. Diagramming the bromodomain interactome network and molecular characterization of these interactions are key to unraveling context-dependent signaling pathways. Herein, we employ a chemoproteomic platform, called interaction-based protein profiling (IBPP), to map the acetylome of the bromodomain and extra terminal domain (BET) family of bromodomains. We developed photo-responsive bromodomain analogues to carry out UV light-induced azide-acetyllysine crosslinking within the recognition cavity of the domain to capture transient interacting partners present in human cells. Subsequent proteomic and biochemical analyses lead to the identification of an array of acetylated interacting factors, which extend the potential function of BET family proteins beyond transcription. We present here eight high-resolution crystal structures of interactome-bound bromodomains that underscore the atypical binding modes and sequence motifs by which BET members recognize an expanded repertoire of acetylated proteins. In addition, we report an acetyllysine-dependent interaction between BRD4 and interleukin enhancer binding factor 3 (ILF3) in human cells, and uncover its role in recruiting transcriptional regulators to the promoter of the Survivin gene, an anti-apoptotic factor overexpressed in a wide range of human neoplasia. Collectively, our work provides a blueprint for engineering bromodomains, establishes IBPP as a robust chemoproteomic tool to characterize bromodomain interactome, and reveals distinctive recognition modes by which such associations may take place in order to modulate signaling pathways. Furthermore, these structural snapshots offer clues to design specific small-molecule inhibitors against the novel BET interactions and paves the path for exploring the biological significance of this newly identified signaling network.

biochemistry↗