Biology subjects

Venditti, V.

Publications and source records attributed to Venditti, V..

4 recordsLinked to original sources

bioRxiv2026

TurboID proximity labeling reveals protein interaction networks for germline Argonautes

In the germline, small RNA-mediated gene regulatory pathways are associated with phase separated condensates known as germ granules across species. Germ granules can be challenging to purify based on their biophysical properties, therefore we used an in vivo proximity labeling approach, TurboID, to define the proximal interaction networks of the germ granule-localized Argonaute proteins: CSR-1, PRG-1/Piwi, PPW-2/WAGO-3, WAGO-1 and WAGO-4, and the nuclear-enriched Argonaute HRDE-1 as a comparator, in C. elegans. We found that although CSR-1 and WAGO-4 bind to the same set of 22G- small RNAs and target an overlapping set of germline genes, they possess distinct proximal interaction networks. Displacement of WAGO-4 from the germ granules highlights distinct interactors involved in chromosome biology and points to a potential nuclear role for WAGO-4. PRG-1/Piwi, WAGO-1, and PPW-2/WAGO-3, act in a common pathway and share an interaction network, while HRDE-1 displays a distinct interaction network that highlights its potential to influence splicing, rather than its widely accepted role in histone modification. Performing TurboID on other sRNA pathway factors, including the RdRP RRF-1 led us to uncover a potential nuclear role for this protein, and overlapping our TurboID data with published proximity labeling data enabled us to identify a core set of eleven mostly uncharacterized proteins that play roles in fertility, germ granule organization, and transgenerational epigenetic inheritance. One of these factors, D2005.4, appears to be a heme-binding germ granule-localized protein with a Tetratricopeptide Repeat Domain that interacts with all germ granule AGOs and may play scaffolding or enzymatic roles in germ granules. Collectively, our work highlights the utility of proximity labelling to uncover insights into AGO and sRNA pathway factor function and identify new factors that could be of importance in germ cell biology.

developmental biology↗

bioRxiv2024

Enhanced identification of small molecules binding to hnRNPA1 via cryptic pockets mapping coupled with X-Ray fragment screening

The human heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a prototypical RNA-binding protein essential in regulating a wide range of post-transcriptional events in cells. As a multifunctional protein with a key role in RNA metabolism, deregulation of its functions has been linked to neurodegenerative diseases, tumour aggressiveness and chemoresistance, which has fuelled efforts to develop novel therapeutics that modulates its RNA binding activities. Here, using a combination of Molecular Dynamics (MD) simulations and graph neural network pockets predictions, we showed that hnRNPA1 N-terminal RNA binding domain (UP1) contains several cryptic pockets capable of binding small molecules. To identify chemical entities for development of potent drug candidates and experimentally validate identified druggable hotspots, we carried out a large fragment screening on UP1 protein crystals. Our screen identified 36 hits which extensively samples UP1 functional regions involved in RNA recognition and binding, as well as mapping hotspots onto novel protein interaction surfaces. We observed a wide range of ligand-induced conformational variation, by stabilisation of dynamic protein regions. Our high-resolution structures, the first of an hnRNP in complex with a fragment or small molecule, provides rapid routes for the rational development of a range of different inhibitors and chemical tools for studying molecular mechanisms of hnRNPA1 mediated splicing regulation.

biochemistry↗

bioRxiv2023

Temperature-Sensitive Contact Modes Allosterically Gate TRPV3

TRPV Ion channels are sophisticated molecular sensors designed to respond to distinct temperature thresholds. The recent surge in cryo-EM structures has provided numerous insights into the structural rearrangements accompanying their opening and closing; however, the molecular mechanisms by which TRPV channels establish precise and robust temperature sensing remain elusive. In this work we employ molecular simulations, multi-ensemble contact analysis, graph theory, and machine learning techniques to reveal the temperature-sensitive residue-residue interactions driving allostery in TRPV3. We find that groups of residues exhibiting similar temperature-dependent contact frequency profiles cluster at specific regions of the channel. The dominant mode clusters on the ankyrin repeat domain and displays a linear melting trend while others display non-linear trends. These modes describe the residue-level temperature response patterns that underlie the channels functional dynamics. With network analysis, we find that the community structure of the channel changes with temperature. And that a network of high centrality contacts connects distant regions of the protomer to the gate, serving as a means for the temperature-sensitive contact modes to allosterically regulate channel gating. Using a random forest model, we show that the contact states of specific temperature-sensitive modes are indeed predictive of the channel gates state. Supporting the physical validity of these modes and networks are several residues identified with our analyses that are reported in literature to be functionally critical. Our results offer high resolution insight into thermo-TRP channel function and demonstrate the utility of temperature-sensitive contact analysis.

biophysics↗

bioRxiv2022

Temperature Sensitive Contacts in Disordered Loops Tune Enzyme I Activity

Homologous enzymes with identical folds often exhibit different thermal and kinetic behaviors. Understanding how enzyme sequence encodes catalytic activity at functionally optimal temperatures is a fundamental problem in biophysics. Recently it was shown that the residues that tune catalytic activities of thermophilic/mesophilic variants of the C-terminal domain of bacterial Enzyme I (EIC) are largely localized within disordered loops, offering a model system with which to investigate this phenomenon. In this work, we employ molecular dynamics simulations and mutagenesis experiments to reveal a mechanism of sequence-dependent activity tuning of EIC homologs. We find that a network of contacts in the catalytic loops is particularly sensitive to changes in temperature, with some contacts exhibiting distinct linear or non-linear temperature-dependent trends. Moreover, these trends define structurally clustered dynamical modes and can distinguish regions that tend toward order or disorder at higher temperatures. Assaying several thermophilic EIC mutants, we show that complementary mesophilic mutations to the most temperature-sensitive positions exhibit the most enhanced activity while mutations to relatively temperature insensitive positions exhibit the least enhanced activities. These results provide a mechanistic explanation of sequence-dependent temperature tuning and offer a computational method for rational enzyme modification. SignificanceTemperature affects the catalytic rates of all enzymes. The impact of temperature on the catalytic activity of an enzyme, however, is convoluted from contributions of protein sequence, structure, and dynamics. As such, understanding and designing the molecular features of enzymes which tune catalytic rates at different temperatures remains a fundamental challenge in biophysics. In this work we have employed molecular simulations and mutagenesis experiments to reveal the temperature tuning mechanism of mesophilic and thermophilic homologues of the C domain of bacterial Enzyme l. We find that enzymes can be tuned to their physiological temperatures through a network of temperature-sensitive residue contacts localized in the disordered loops. Furthermore, we find that among temperature-sensitive contacts some exhibit linear and others non-linear dependence on temperature. These clues offer a promising physics-based approach for tuning enzyme activity.

biophysics↗