Biology subjects

Lama, D.

Publications and source records attributed to Lama, D..

11 recordsLinked to original sources

bioRxiv2026

Single-molecule mass measurements uncover shifting RNA interactions during condensate phase transitions

RNA interactions are a key contributor to the formation and disassembly of intracellular protein condensates. Although some proteins utilize specific RNA-binding domains, these processes can also be mediated by charge interactions with intrinsically disordered regions. Due to the dynamic nature of these systems, investigating the underlying specificity and stoichiometry remains challenging. Here, we demonstrate that single-molecule mass measurements with mass photometry can capture RNA-protein interactions in phase-separated protein systems. Using the approach to investigate RNA-mediated phase shifts of tau condensates, we find that increasing the RNA concentration, which promotes phase re-entry, results in RNA-mediated tau multimerization, where each tau monomer binds a linear RNA sequence of approximately 30 nucleotides. Solution NMR and native mass spectrometry confirm the formation of stable complexes between RNA and the basic proline-rich and repeat domains of tau, which have a net charge of -29. Our findings demonstrate that mass photometry can distinguish between charge neutralization, which drives coacervation, and complex formation, which mediates phase re-entry, making it a highly complementary tool for the study of RNA-mediated phase separation.

biophysics↗

bioRxiv2026

A stapled peptide inhibitor of MDM2 enables pharmacological activation of p53 in zebrafish

Measuring the activity of the tumor suppressor p53 in living systems is essential for understanding its dysregulation in cancer and other conditions, such as aging and diabetes. Zebrafish (Danio rerio) are a powerful vertebrate model that enable such studies, due to the evolutionary conservation of p53 structure and function. However, p53 activity in zebrafish has mainly been assessed using pharmacological methods that induce DNA damage or have off-target effects, making it difficult to isolate p53-specific responses from broader stress responses. Here, by using biophysical assays, molecular dynamics, and molecular assays, we show that sulanemadlin, a stapled peptide inhibitor of MDM2, binds to zebrafish Mdm2 and transcriptionally activates downstream targets of p53, including cdkn1a, isoform{Delta} 113p53, and Mdm2. No effect on gene expression was observed in embryos treated with a point-modified control peptide or in embryos carrying a mutation that renders p53 transcriptionally inactive. RNA sequencing further confirmed upregulation of p53 signaling and downregulation of DNA replication pathways, while an acridine orange assay showed no detectable increases in apoptosis. In contrast, the tested small molecule Mdm2 inhibitors exhibit reduced binding affinity to zebrafish Mdm2 due to an amino acid variation in the zebrafish Mdm2 binding pocket. By overcoming a species-specific barrier in p53-MDM2 binding, the stapled peptide sulanemadlin is the first pharmacological tool to specifically activate p53 in zebrafish without inducing measurable apoptosis, enabling direct in vivo studies of p53 regulation in cancer and other disease contexts.

pharmacology and toxicology↗

bioRxiv2026

Liquid-liquid phase separation of tau regulates client binding via a conformational relay

Intrinsically disordered proteins (IDPs) achieve functional specificity through dynamic, multivalent interactions that are often reorganized during liquid-liquid phase separation (LLPS). How LLPS reshapes IDP conformational landscapes and binding preferences remains poorly understood. Here, we combine native ion mobility mass spectrometry, mass photometry, fluorescence microscopy, and computational modeling to dissect how conditions that promote LLPS of the intrinsically disordered human tau protein regulate its interactions with tubulin and the neuronal anti-amyloid chaperone domain BRICHOS from Bri2. We show that electrostatically driven compaction and transient oligomerization of tau during LLPS promote BRICHOS binding to a specific Tau segment. Within tau condensates, BRICHOS can compete with tubulin for tau interactions by blocking its adjacent binding site, thereby modulating LLPS-dependent microtubule assembly. Using a generalizable experimental strategy, we provide a proof of concept for detecting conformational selectivity in a dynamic condensate.

biophysics↗

bioRxiv2023

Adrenergic Reprogramming of Preexisting Adipogenic Trajectories Steer Naive Mural Cells Toward Beige Differentiation

In adult white adipose tissue, cold or {beta}3-adrenoceptor activation promotes the appearance of thermogenic beige adipocytes. Our comprehensive single-cell analysis revealed that these cells arise through the reprogramming of existing adipogenic trajectories, rather than from a single precursor. These trajectories predominantly arise from SM22-expressing vascular mural progenitor cells. Central in this transition is the activation of Adrb3 in mature adipocytes, leading to subsequent upregulation of Adrb1 in primed progenitors. Under thermoneutral conditions, synergistic activation of both Adrb3 and Adrb1 recapitulates the pattern of cold-induced SM22+ cell recruitment. Lipolysis-derived eicosanoids, specifically docosahexaenoic acid (DHA) and arachidonic acid (AA) prime these processes and in vitro, were sufficient to recapitulate progenitor cells priming. Collectively, our findings provide a robust model for cold-induced beige adipogenesis, emphasizing a profound relationship between mature adipocytes and mural cells during cold acclimation, and revealing the metabolic potential of this unique cellular reservoir.

cell biology↗

bioRxiv2022

Mass spectrometry of RNA-binding proteins during liquid-liquid phase separation reveals distinct assembly mechanisms and droplet architectures

Phase separation of heterogeneous ribonucleoproteins (hRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility-mass spectrometry, and molecular dynamics simulations. We used a phase separation-compatible spider silk domain and pH changes to control the self-assembly of the hRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with phase separation. We find that NT*-FUS monomers undergo an unfolded-to-globular transition, whereas NT*-TDP-43 oligomerizes into partially disordered dimers and trimers. NT*-hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over phase separation. The divergent assembly mechanisms result in structurally distinct complexes, indicating differences in RNA processing and translation depending on biological context.

biophysics↗

bioRxiv2022

A grappling hook interaction balances self-assembly and chaperone activity of Nucleophosmin 1

How the self-assembly of partially disordered proteins generates functional compartments in the cytoplasm and particularly in the nucleus is poorly understood. Nucleophosmin 1 (NPM1) is an abundant nucleolar protein that forms large oligomers which provide the scaffold for ribosome assembly but also prevent protein aggregation as part of the cellular stress response. Examining the relationship between the self-assembly and chaperone activity of NPM1, we find that oligomerization of full-length NPM1 modulates its ability to retard amyloid formation in vitro. Machine learning and cryo-electron microscopy reveal fuzzy interactions between the disordered region and the C-terminal nucleotide-binding domain that cross-link NPM1 pentamers into oligomers. Ribosomal peptides mediate in a tighter association within the oligomers, reducing their capacity to prevent amyloid formation. We conclude that NPM1 uses a "grappling hook" interaction to form a network-like structure whose chaperone activity is tuned by basic proteins, suggesting a regulatory mechanism for the nucleolar stress response.

biophysics↗

bioRxiv2022

Development of a Novel Peptide Aptamer that Interacts with the eIF4E Capped-mRNA Binding Site using Peptide Epitope Linker Evolution (PELE).

Identifying new binding sites and poses that modify biological function are an important step towards drug discovery. We have identified a novel disulphide constrained peptide that interacts with the cap-binding site of eIF4E, an attractive therapeutic target that is commonly overexpressed in many cancers and plays a significant role in initiating a cancer specific protein synthesis program though binding the 5cap (7methyl-guanoisine) moiety found on mammalian mRNAs. The use of disulphide constrained peptides to explore intracellular biological targets is limited by their lack of cell permeability and the instability of the disulphide bond in the reducing environment of the cell, loss of which results in abrogation of binding. To overcome these challenges, the cap-binding site interaction motif was placed in a hypervariable loop on an VH domain, and then selections performed to select a molecule that could recapitulate the interaction of the peptide with the target of interest in a process termed Peptide Epitope Linker Evolution (PELE). A novel VH domain was identified that interacted with the eIF4E cap binding site with a nanomolar affinity and that could be intracellularly expressed in mammalian cells. Additionally, it was demonstrated to specifically modulate eIF4E function by decreasing cap-dependent translation and cyclin D1 expression, common effects of eIF4F complex disruption.

biochemistry↗

bioRxiv2021

A spindle and thread-mechanism unblocks translation of N-terminally disordered proteins

Protein disorder is a major hurdle for structural biology. A prominent example is the tumour suppressor p53, whose low expression levels and poor conformational stability due to a high degree of disorder pose major challenges to the development of cancer therapeutics. Here, we address these issues by fusing p53 to an engineered spider silk domain termed NT*. The chimeric protein displays highly efficient translation in vitro and in E. coli and is fully active in human cancer cells. The transmission electron microscopy structure and native mass spectrometry reveal that the full-length p53 fusion protein adopts a compact conformation. Molecular dynamics simulations show that the disordered transactivation domain of p53 is wound around the NT* domain via a series of folding events, resulting in a globular structure. We find that expression of B-Raf, another partially disordered cancer target, is similarly enhanced by fusion to NT*. In summary, we demonstrate how inducing co-translational folding via a molecular "spindle and thread" mechanism can overcome poor translation efficiency of partially disordered proteins.

biochemistry↗

bioRxiv2020

TRAF4 inhibits bladder cancer progression by promoting BMP/SMAD signalling pathway

Bladder cancer is one of the most prevalent cancer types in the world, frequently exhibiting invasion and metastasis and therefore associated with poor prognosis. It is a progressive disease with high recurrence rates even after surgery, which calls for the urgent need for early intervention and diagnosis. The E3 ubiquitin ligase TNF Receptor Associated Factor 4 (TRAF4) has been largely implicated as a tumour-promoting element in a wide range of cancers. Over-expression and amplification of this gene product has been a common observation in breast and other metastatic tumours. In contrast, we observed that expression of TRAF4 negatively correlated with overall patient survival. Moreover, its expression was repressed at epigenetic levels in aggressive bladder cancer cells. We also describe an ERK kinase phosphorylation site on TRAF4 that inhibits its stability and localization to plasma membrane in such cells. Furthermore, knockdown of TRAF4 in epithelial bladder cancer cell lines leads to gain of mesenchymal genes and a loss of epithelial integrity. Reciprocally, stable over-expression of TRAF4 in mesenchymal cells leads to decreased migratory and invasive properties. Transcriptomic analysis upon TRAF4 mis-expression in bladder cancer cell lines revealed that higher TRAF4 expression enhanced BMP/SMAD and dampened NF-{kappa}B signalling pathways. Importantly, this notion was confirmed in bladder cancer patient material. Mechanistically, we showed that TRAF4 targets the E3 ubiquitin ligase SMURF1, a negative regulator of BMP/SMAD signalling, for proteasomal degradation in bladder cancer cells. We show that genetic and pharmacological inhibition of SMURF1 or its function inhibited migration of aggressive (mesenchymal) bladder cancer cells.

cancer biology↗

bioRxiv2020

Conformational ordering of intrinsically disordered peptides for targeting translation initiation

Intrinsically disordered regions (IDRs) in proteins can regulate their activity by facilitating protein-protein interactions (PPIs) as exemplified in the recruitment of the eukaryotic translation initiation factor 4E (eIF4E) protein by the protein eIF4G. Deregulation of this PPI module is central to a broad spectrum of cancer related malignancies and its targeted inhibition through bioactive peptides is a promising strategy for therapeutic intervention. We have employed a structure-guided approach to rationally develop peptide derivatives from the intrinsically disordered eIF4G scaffold by incorporating non-natural amino acids that facilitates disorder-to-order transition. The conformational heterogeneity of these peptides and the degree of structural reorganization required to adopt the optimum mode of interaction with eIF4E underscores their differential binding affinities. The presence of a pre-structured local helical element in the ensemble of structures was instrumental in the efficient docking of the peptides on to the protein surface. These insights were exploited to further design features into the peptide to propagate bound-state conformations in solution which resulted in the generation of a potent eIF4E binder. The study illustrates the molecular basis of eIF4E recognition by a disordered epitope from eIF4G and its modulation to generate peptides that can potentially attenuate translation initiation in oncology.

biophysics↗

bioRxiv2018

Phosphorylation regulates auto-inhibition of kinesin KIF3A

Kinesin are molecular motors that move along the microtubules. They function to transport cargoes, vesicles and organelles to designated locations in the cells. KIF3A belongs to the Kinesin-2 family and forms a heterotrimeric complex with KIF3B and KAP3. We have earlier shown that the cargo trafficking activity of KIF3 motor can be regulated by CaMKII kinase and POPX2 phosphatase. In this study, we elucidated the mechanism of KIF3A regulation. We find that KIF3A adopts an auto-inhibited state through the interaction between the motor and tail domains. The motor-tail interaction also hinders the ATPase activity of the motor domain. We show that the phosphorylation status of serine-689/690 (mouse/human) at the C-terminal region of KIF3A is crucial for the motor-tail interaction. The motor domain does not interact well with the tail domain when serine-689 is phosphorylated by CaMKII or mutated to aspartic acid to mimic phosphorylation. Molecular dynamics simulations suggest that the non-phosphorylated tail domain folds into the hydrophobic pocket formed by the motor dimers. Phosphorylation of serine-689 results in conformational changes that leads to the relieve of auto-inhibition.

biochemistry↗