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Bah, A.

Publications and source records attributed to Bah, A..

11 recordsLinked to original sources
bioRxiv2026

Phosphoserine clusters as metal ion sensors for protein phase separation

Phosphorylation is a major regulator of biomolecular condensation, yet it remains unclear whether clustered phosphoserines can directly tune phase behavior via metal-ion coordination. Here, using solution NMR spectroscopy and human heterochromatin protein 1 (HP1) as a model system, we show that stepwise phosphorylation of its N-terminal serine cluster generates a dynamic metal-responsive module that engages Mg{superscript 2}, Ca{superscript 2}, and Mn{superscript 2}, whereas the unmodified protein shows little or no response. Metal coordination lowers the saturation concentration of phosphorylated HP1, reshapes the temperature-dependent stability of its condensates, and modulates the effects of peptide regulators in an ion-specific manner. Our data support a model in which weak, transient metal-mediated contacts enhance intermolecular connectivity between phosphorylated HP1 molecules, promoting reversible condensation alongside canonical electrostatic interactions. These findings establish clustered phosphoserines as sequence-encoded metal-responsive elements that couple post-translational modification to the material properties of biomolecular condensates.

biophysics↗
bioRxiv2026

Lipoengineering of Biomolecular Condensates Controls Material Properties and Multiphase Hierarchy to Guide Organoid Morphogenesis

Cells use post-translational modifications (PTMs) to reconfigure biomolecular condensates across length scales, space, and time.1,2 While charged PTMs are well-known electrostatic switches,3,4 how ubiquitous neutral PTMs shape condensate plasticity and hierarchy remains unclear. Here, we establish a set of design principles for using site-specific lipidation, a class of neutral hydrophobic PTMs, to rationally control properties and interactions of engineered biomolecular condensates. Through systematic analysis of over 80 lipidated synthetic intrinsically disordered proteins (IDPs), we uncovered two distinct axes of control. First, the interplay between the lipid and the local three-residue sequence of its attachment site acts as a programmable switch for cohesion--the homotypic interactions that define the material state of the condensed phase-- directing assemblies toward dynamic liquids, arrested gels, or ordered fibrillar solids. Second, the lipid, together with the global properties of the IDP scaffold, tunes adhesion--the heterotypic interactions that govern condensate miscibility and hierarchical organization. We harnessed these principles to rationally engineer complex, multi-phase architectures and create hybrid hydrogels with programmed microstructure and material properties that guide the morphogenesis of functional intestinal organoids. These findings establish a new framework for lipoengineering advanced biomaterials and provide a blueprint for dissecting structure-property relationships across diverse classes of PTMs.

bioengineering↗
bioRxiv2024

Uncovering the molecular interactions underlying MBD2 and MBD3 phase separation

Chromatin organization controls DNAs accessibility to regulatory factors to influence gene expression. Heterochromatin, or transcriptionally silent chromatin enriched in methylated DNA and methylated histone tails, self-assembles through multivalent interactions with its associated proteins into a condensed, but dynamic state. Liquid-liquid phase separation (LLPS) of key heterochromatin regulators, such as heterochromatin protein 1 (HP1), plays an essential role in heterochromatin assembly and function. Methyl-CpG-binding protein 2 (MeCP2), the most studied member of the methyl-CpG-binding domain (MBD) family of proteins, has been recently shown to undergo LLPS in the absence and presence of methylated DNA. These studies provide a new mechanistic framework for understanding the role of methylated DNA and its readers in heterochromatin formation. However, the details of the molecular interactions by which other MBD family members undergo LLPS to mediate genome organization and transcriptional regulation are not fully understood. Here, we focus on two MBD proteins, MBD2 and MBD3, that have distinct but interdependent roles in gene regulation. Using an integrated computational and experimental approach, we uncover the homotypic and heterotypic interactions governing MBD2 and MBD3 phase separation and DNAs influence on this process. We show that despite sharing the highest sequence identity and structural homology among all the MBD protein family members, MBD2 and MBD3 exhibit differing residue patterns resulting in distinct phase separation mechanisms. Understanding the molecular underpinnings of MBD protein condensation offers insights into the higher-order, LLPS-mediated organization of heterochromatin.

biochemistry↗
bioRxiv2023

Delineating Structural Propensities of the 4E-BP2 Protein via Integrative Modelling and Clustering

The intrinsically disordered 4E-BP2 protein regulates mRNA cap-dependent translation through the interaction with the predominantly folded eukaryotic initiation factor 4E (eIF4E). Phosphorylation of 4E-BP2 dramatically reduces eIF4E binding, in part by stabilizing a binding- incompatible folded domain (REF). Here, we used a Rosetta-based sampling algorithm optimized for IDRs to generate initial ensembles for two phospho forms of 4E-BP2, non- and five-fold phosphorylated (NP and 5P, respectively), with the 5P folded domain flanked by N- and C-terminal IDRs (N-IDR and C-IDR, respectively). We then applied an integrative Bayesian approach to obtain NP and 5P conformational ensembles that agree with experimental data from nuclear magnetic resonance, small-angle X-ray scattering and single-molecule Forster resonance energy transfer (smFRET). For the NP state, inter-residue distance scaling and 2D maps revealed the role of charge segregation and pi interactions in driving contacts between distal regions of the chain ([~]70 residues apart). The 5P ensemble shows prominent contacts of the N-IDR region with the two phosphosites in the folded domain, pT37 and pT46, and, to a lesser extent, delocalized interactions with the C-IDR region. Agglomerative hierarchical clustering led to partitioning of each of the two ensembles into four clusters, with different global dimensions and contact maps. This helped delineate an NP cluster that, based on our smFRET data, is compatible with the eIF4E-bound state. 5P clusters were differentiated by interactions of C-IDR with the folded domain and of the N-IDR with the two phosphosites in the folded domain. Our study provides both a better visualization of fundamental structural poses of 4E-BP2 and a set of falsifiable insights on intrachain interactions that bias folding and binding of this protein.

biophysics↗
bioRxiv2023

ABI1 regulates transcriptional activity of Androgen Receptor by novel DNA and AR binding mechanism

Transcription regulates key functions of living organisms in normal and disease states, including cell growth and development, embryonic and adult tissue organization, and tumor progression. Here we identify a novel mechanism of transcriptional regulation by an actin regulatory and signaling protein, Abelson Interactor 1 (ABI1). Using prostate cancer models, we uncover a reciprocal regulation between ABI1 and the Androgen Receptor (AR). ABI1 is a direct, androgen-regulated target; in turn, ABI1 interacts with AR and its splice variant ARv7, and co-regulates a subset of specific transcriptional targets. ABI1 directs transcription through transient yet well-defined interaction of its intrinsically disordered region with DNA. Clinical evaluation shows that the ABI1-DNA binding (through Exon 4 splicing) and ABI1-AR interaction are regulated during androgen deprivation therapy and prostate cancer progression, thus controlling tumor plasticity through connecting actin cytoskeleton and cellular signaling to transcriptional regulation. We propose ABI1 as epigenetic regulator of transcriptional homeostasis in AR-driven cancers. Statement of importanceThis study describes fundamental discovery in prostate cancer identifying novel mechanism of transcription by unique DNA binding mechanism involving actin cytoskeleton regulatory protein ABI1. ABI1-DNA binding activity predicts survival of prostate cancer patients. Moreover, we discover ABI1-AR reciprocal regulation that has far reaching implications for tumor plasticity and androgen-sensitive pathogenesis.

cancer biology↗
bioRxiv2023

The post-translational modification profile of TAR DNA-Binding Protein (TDP-43) in platelets of patients with Alzheimer's disease: An exploratory study for blood-based biomarker development.

The assignment of blood-based biomarkers for neurodegenerative diseases is of great clinical value. Well-developed and validated blood-based biomarkers can serve in early diagnosis and prognosis as well as aid in patient screening when recruiting for clinical trials. We attempted to establish a portfolio for post-translationally modified TAR DNA/RNA-binding protein (TDP-43), a regulator of nuclear transcription, in platelet cytosol obtained from patients with Alzheimers disease (AD) comparing to age-matched healthy subjects and a disease control cohort. We aimed to identify the most prominent post-translational modifications of TDP-43 as an AD-relevant biomarker and to demonstrate that such an assessment can be performed in peripheral blood. We have isolated TDP-43 protein from human platelet cytosol utilizing an Immunoaffinity chromatography. The eluates were immunoprobed with a series of antibodies raised against post-translationally modified proteins. We employed a capillary electrophoretic immunoassay (CEI) to assess the phosphorylated TDP-43 profile. We observed that SUMOylation, phosphorylation, ubiquitination, and cysteine oxidation of TDP-43 are more prominent in platelet cytosol of AD patients as compared to control subjects. These studies will pave the way for identifying disease-specific TDP-43 derivatives that can be potential biomarkers for early diagnosis and the development of therapeutics.

neuroscience↗
bioRxiv2022

Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock

Circadian clocks are composed of molecular oscillators that pace rhythms of gene expression to the diurnal cycle. Therein, transcriptional-translational negative feedback loops (TTFLs) generate oscillating levels of transcriptional repressor proteins that regulate their own gene expression. In the filamentous fungus Neurospora crassa, the proteins Frequency (FRQ), the FRQ-interacting RNA helicase (FRH) and Casein-Kinase I (CK1) form the FFC complex that represses expression of genes activated by the White-Collar complex (WCC). A key question concerns how FRQ orchestrates molecular interactions at the core of the clock despite containing little predicted tertiary structure. We present the reconstitution and biophysical characterization of FRQ and the FFC in unphosphorylated and highly phosphorylated states. Site-specific spin labeling and pulse- dipolar ESR spectroscopy provides domain-specific structural details on the full-length, 989- residue intrinsically disordered FRQ and the FFC. FRQ contains a compact core that associates and organizes FRH and CK1 to coordinate their roles in WCC repression. FRQ phosphorylation increases conformational flexibility and alters oligomeric state but the changes in structure and dynamics are non-uniform. Full-length FRQ undergoes liquid-liquid phase separation (LLPS) to sequester FRH and CK1 and influence CK1 enzymatic activity. Although FRQ phosphorylation favors LLPS, LLPS feeds back to reduce FRQ phosphorylation by CK1 at higher temperatures. Live imaging of Neurospora hyphae reveals FRQ foci characteristic of condensates near the nuclear periphery. Analogous clock repressor proteins in higher organisms share little position-specific sequence identity with FRQ; yet, they contain amino-acid compositions that promote LLPS. Hence, condensate formation may be a conserved feature of eukaryotic circadian clocks.

biochemistry↗
bioRxiv2022

Multisite Phosphorylation and Binding Alter Conformational Dynamics of the 4E-BP2 Protein

Intrinsically disordered proteins (IDPs) play critical roles in regulatory protein interactions, but detailed structural/dynamics characterization of their ensembles remain challenging, both in isolation and they form dynamic fuzzy complexes. Such is the case for mRNA cap-dependent translation initiation, which is regulated by the interaction of the predominantly folded eukaryotic initiation factor 4E (eIF4E) with the intrinsically disordered eIF4E binding proteins (4E-BPs) in a phosphorylation-dependent manner. Single-molecule Forster resonance energy transfer showed that the conformational changes of 4E-BP2 induced by binding to eIF4E are non-uniform along the sequence; while a central region containing both motifs that bind to eIF4E expands and becomes stiffer, the C-terminal region is less affected. Fluorescence anisotropy decay revealed a nonuniform segmental flexibility around six different labelling sites along the chain. Dynamic quenching of these fluorescent probes by intrinsic aromatic residues measured via fluorescence correlation spectroscopy report on transient intra- and inter-molecular contacts on ns-s timescales. Upon hyperphosphorylation, which induces folding of ~40 residues in 4E-BP2, the quenching rates decreased at most labelling sites. The chain dynamics around sites in the C-terminal region far away from the two binding motifs significantly increased upon binding to eIF4E, suggesting that this region is also involved in the highly dynamic 4E-BP2:eIF4E complex. Our time-resolved fluorescence data paint a sequence-level rigidity map of three states of 4E-BP2 differing in phosphorylation or binding status and distinguish regions that form contacts with eIF4E. This study adds complementary structural and dynamics information to recent studies of 4E-BP2, and it constitutes an important step towards a mechanistic understanding of this important IDP via integrative modelling.

biophysics↗
bioRxiv2021

FMRP bridges R-loops and DHX9 through direct interactions

Mutations in, or deficiency of, FMRP is responsible for the Fragile X syndrome (FXS), the most common cause for inherited intellectual disability. FMRP is a nucleocytoplasmic protein, primarily characterized as a translation repressor with poorly understood nuclear function(s). We recently uncovered a genome protective role of FMRP. We reported that FXS patient-derived cells lacking FMRP sustain higher level of DNA double-strand breaks than normal cells, a phenotype further exacerbated by DNA replication stress. The stress-induced DSBs occur at sequences prone to form R-loops, which are co-transcriptional RNA:DNA hybrids that have been associated with genome instability. Concordantly, we showed that FXS cells accumulate R-loops under replication stress. Moreover, expression of FMRP and not a mutant deficient in binding nucleic acids and known to cause FXS, FMRPI304N, reduced R-loop-associated DSBs. These observations demonstrated that FMRP promotes genome integrity by preventing R-loop accumulation and chromosome breakage. Here, we explore the mechanism through which FMRP prevents R-loop accumulation in an isogenically controlled CRISPR KO of FMR1 (gene encoding for FMRP) in HEK293T cells. We demonstrate for the first time that FMRP directly binds R-loops. We show that FMRP interacts with DHX9, an RNA helicase that unwinds both double strand RNA and RNA:DNA hybrids and regulates R-loop formation through modulating these activities. This interaction is reduced with FMRPI304N, suggesting that FMRP regulation of R-loop is mediated through DHX9. Interestingly, we show that FMRP inhibits DHX9 helicase activity on RNA:DNA hybrids. Moreover, DHX9 binds chromatin containing R-loops more efficiently in the absence of a functional FMRP. These results suggest an antagonistic relationship between FMRP and DHX9 at the chromatin, where FMRP prevents R-loop formation by suppressing DHX9. Our study sheds new light on our understanding of the genome functions of FMRP.

biochemistry↗
bioRxiv2020

Inhibition of ITK differentiates GVT and GVHD in allo-HSCT

Allogeneic hematopoietic stem cell transplantation is a life-saving treatment for many malignant and nonmalignant diseases. Donor T cells contained within the graft prevent tumor recurrence via graft-versus-tumor (GVT) effects, however, also cause graft-versus-host disease (GVHD). Novel treatment strategies are therefore needed to allow maintenance of GVT while suppressing GVHD. Here we show using murine models, that targeting IL-2-inducible T cell kinase (ITK) in donor T cells reduces GVHD while preserving the beneficial GVT effects. Donor T cells from Itk-/- mice exhibit significantly reduced production of inflammatory cytokines and migration to GVHD target organs such as liver and small intestine, while maintaining GVT efficacy against primary B-ALL tumors. Itk-/- T cells exhibited reduced expression of IRF4 and decreased JAK/STAT signaling activity, but preserved cytotoxicity, which was accompanied by upregulation of Eomesodermin (Eomes), which was necessary for GVT function. A novel peptide inhibitor ITK signaling is also able to prevent GVHD. This novel peptide inhibitor also reduced cytokine production in mice and human T cells. Altogether, our data suggest that inhibiting ITK could be a therapeutic strategy to reduce GVHD while preserving the beneficial GVT effects following allo-HSCT treatment. Key PointsO_LIInhibiting ITK by a novel peptide significantly reduces GVHD but retains GVT. C_LIO_LIITK deficient donor T cells exhibit minimal GVHD, but maintain GVT activity. C_LIO_LIITK deficient donor T cells exhibit significantly reduced production of inflammatory cytokines and migration to GVHD target organs. C_LIO_LIEomes is required for GVT effect. C_LI

immunology↗