Biology subjects

Cherfils, J.

Publications and source records attributed to Cherfils, J..

6 recordsLinked to original sources

bioRxiv2025

Mapping Bidirectional Allosteric Communication in Arf GTPases

The Arf GTPases are eukaryotic signaling proteins implicated in trafficking, motility and membrane remodeling. They must undergo a massive conformational transition in the switch between their inactive, GDP-bound form and their GTP-bound form, competent for downstream signaling. The mechanism of their GDP-to-GTP nucleotide switch implicates a functional molten globule (MG) ensemble. Access to this ensemble and apparent spontaneous switching is modulated by residues in the C-terminal half of the different homologs through a back-to-front allosteric pathway. Here, using high pressure (HP) NMR we show that a mutation in the N-terminal switch region in the front of Arf1 known to modulate spontaneous switching perturbs the stability of residues on a path that stretches from the front to the back. This establishes the existence of a bidirectional and continuous allosteric pathway that passes through the GDP ligand. HP switching studies also demonstrate that the allosteric mechanism controls access to the functional MG state, rather than directly affecting the switching rates. Secondary AbstractThe mechanism of the Arf GTPase nucleotide switch implicates a functional molten globule (MG) ensemble. Switching by the front side of the Arf proteins is controlled by sequences on the back side. Here, using high pressure (HP) NMR we map a bidirectional and continuous allosteric pathway that passes between front and back through the GDP ligand. We also demonstrate that the allosteric mechanism controls access to the functional MG state, rather than directly affecting the switching rates.

biophysics↗

bioRxiv2025

Putting a lid on it: The N-terminal helix of Arf1 inhibits switching via stabilization of the overall native state

The Arf (and Arf-like) GTPases, unlike all other Ras family GTPase members, exhibit a repressed conformation in the inactive, GDP-bound form. The N-terminal helix of Arf GTPases, which is missing in the other Ras family members, caps the switch region, confining it to this repressed state. Nucleotide exchange and activation involve a massive conformational change made possible by the dissociation of the N-terminal helix from the core of the protein. Spontaneous switching in Arfs is enhanced upon deletion of this helix. While the structural and functional role of the N-terminal helix in Arf proteins is well-known, the energetic basis for its effects have not been established. Here we mapped the local stability of the Arf1{Delta}17 variant, deleted for the N-terminal helix, using high pressure biophysical approaches and compared it to that of full-length Arf1. Deletion of the N-terminal helix decreased Arf1 stability across the entire structure. Thus, rather than imposing a specific structural pathway for repression, the N-terminal helix exercises global control of Arf1 stability to repress switching. Statement of SignificanceEnergetic mapping of the N-terminal deletion mutant of Arf1 using high pressure biophysical approaches reveals that this helix maintains the protein in the repressed state unique to Arf and Arf-like GTPases via overall stabilization of the native state rather than unique allosteric communication with the switch region.

biophysics↗

bioRxiv2021

Arpin regulates migration persistence by interacting with both tankyrases and the Arp2/3 complex

During cell migration, protrusion of the leading edge is driven by the polymerization of Arp2/3-dependent branched actin networks. Migration persistence is negatively regulated by the Arp2/3 inhibitory protein Arpin. To better understand Arpin regulation in the cell, we looked for interacting partners and identified both Tankyrase 1 and 2 (TNKS) using a yeast two hybrid screen and co-immunoprecipitation with full-length Arpin as a bait. Arpin interacts with ankyrin repeats of TNKS through a C-terminal binding site on its acidic tail overlapping with the Arp2/3 binding site. To uncouple the interactions of Arpin with TNKS and Arp2/3, we introduced point mutations in the Arpin tail and attempted to rescue the increased persistence of the Arpin knock-out using random plasmid integration or compensating knock-in at the ARPIN locus. Arpin mutations impairing either Arp2/3- or TNKS-interaction were insufficient to fully abolish Arpin activity. Only the mutation that affects both interactions rendered Arpin completely inactive, suggesting the existence of two independent pathways, by which Arpin controls migration persistence. Arpin was found to dissolve liquid-liquid phase separation of TNKS upon overexpression. Together these data suggest that TNKS might be mediating the function of Arpin rather than regulating Arpin.

cell biology↗

bioRxiv2019

A toxic palmitoylation on Cdc42 drives a severe autoinflammatory syndrome

BackgroundAutoinflammatory diseases (AID) result from dysregulation of the first lines of innate immune responses. Recently, development of high throughput genome sequencing technology led to the rapid emergence of important knowledge in the genetic field. About 20 genes have been identified so far in monogenic forms of distinct AID. However, 70-90 % of patients with AID remain without genetic diagnosis.\n\nObjectiveWe report the identification and characterization of a mutation in the C-terminal region of the Rho GTPase Cdc42 in a patient presenting a severe autoinflammatory phenotype.\n\nMethodsWe have analyzed the consequences of the mutation on the subcellular localization of the Cdc42 protein using imaging techniques. Molecular studies were performed using proteomic and biochemical experiments to provide mechanistic bases of the observed defects. Functional assays were also conducted using flow cytometry and cytokine production measurements.\n\nResultsWe show that mutant Cdc42 is trapped in the Golgi apparatus due to the aberrant addition of a palmitate that both enhances the interaction of mutant Cdc42 with Golgi membranes and inhibit its extraction by GDP dissociation inhibitor (GDI), thus impairing its cytosol/membrane shuttling. At the functional level, mutant Cdc42 fails to sustain actin filaments polymerization and induces an exacerbated profile of pro-inflammatory cytokine production due to increased NF-{kappa}B activation.\n\nConclusionsOur study now provides a molecular explanation for mutations that have been identified recently in our AID patient and others in the C-terminal part of Cdc42. Mutations located in this region of Cdc42 impair the intracellular localization of Cdc42, preventing its interaction with the plasma membrane. Thus, our results definitively link mutations in the CDC42 gene to a complex immune-hemato-autoinflammatory phenotype in humans.

genetics↗

bioRxiv2019

MglA functions as a three-state GTPase to control movement reversals of Myxococcus xanthus

In Myxococcus xanthus, directed movement is controlled by inter-dependent pole-to-pole oscillations of the small GTPase MglA, its GAP MglB and the RomR protein. However, these proteins have strikingly different oscillatory regimes such that MglA is segregated from MglB and RomR at reversal activation. The molecular mechanism whereby information is exchanged between the lagging and leading poles resulting in MglA detachment from the leading pole during reversals has remained unknown. Here, we show that MglA has two GTP-bound forms, one of which is insensitive to MglB (MglA-GTP*) and is re-sensitized to MglB by a feedback mechanism operated by MglA-GDP. By identifying the region of MglB that is critical for its association to the lagging pole, we demonstrate that MglA-GTP* is functional in vivo. These data suggest that MglA-GDP acts as a soluble messenger to convert polar MglA-GTP* into a diffusible MglA-GTP species, explaining MglA re-localization to the opposite pole during reversals.

microbiology↗

bioRxiv2018

A metal-regulated AMPylation/deAMPylation switch in FIC proteins

FIC proteins regulate molecular processes in bacteria and animals by carrying out various post-translational modifications (PTM) of proteins by phosphate-containing compounds. The most frequent FIC-catalyzed PTM is the addition of AMP using ATP as a cofactor, a reaction coined AMPylation. In a large subgroup of FIC proteins, AMPylation is inhibited by a structurally conserved glutamate, but a diffusible signal able to relieve autoinhibition has not been identified. Here, we addressed this issue by studying two members of this subgroup, a single-domain FIC protein from the bacterial pathogen Enterococcus faecalis (EfFIC) and human HYPE/FicD, which is involved in the unfolded protein response in the endoplasmic reticulum. By combining structural and biochemical analysis, we find that EfFIC catalyzes both AMPylation and deAMPylation, and that both enzymatic activities are borne by the same active site. Remarkably, the conserved glutamate implements a multi-position metal switch, whereby different metals support or inhibit each of these reactions. As a result, the balance between the AMPylation and deAMPylation activities of EfFIC is controlled by the Mg2+/Ca2+ ratio, with Ca2+ favoring deAMPylation. Furthermore, we show that deAMPylation of the endoplasmic reticulum BIP chaperone by human FicD/HYPE is dependent on the Mg2+/Ca2+ ratio, with high Ca2+ concentration impairing deAMPylation. Our findings suggest that the conserved glutamate is a signature of AMPylation/deAMPylation bifunctionnality in FIC proteins. They also identify for the first time a diffusible signal that can rapidly modulate these opposing activities, which opens important perspectives for their functions in bacterial stress and human ER homeostasis.

biochemistry↗