Biology subjects

Gehin, C.

Publications and source records attributed to Gehin, C..

7 recordsLinked to original sources

bioRxiv2025

Molecular Regulation and Physiological Role of 1 GOLPH3-mediated Golgi retention

The Golgi complex serves as the central hub of the biosynthetic pathway, where anterograde and retrograde trafficking converge. How cargo and Golgi-resident proteins traverse this organelle has long been debated. Recent studies have identified a molecular machinery that sorts resident proteins into retrograde-directed COPI vesicles during cisternal maturation. Golgi phosphoprotein 3 (GOLPH3) is a key component of this system; however, its physiological relevance and regulatory mechanisms remain poorly defined. Here, we show that GOLPH3 depletion in mice disrupts both protein and lipid glycosylation, causes partially penetrant embryonic lethality, and severely impairs growth and bone mineralization. At the molecular level, we find that GOLPH3 is regulated by functionally antagonistic S-acylation events that control the topology of its membrane association. To mediate retrograde trafficking of Golgi-resident glycosyltransferases, GOLPH3 must bind their cytosolic tails. This occurs via a negatively charged surface region, which is correctly oriented only in one of the S-acylated GOLPH3 conformations. Together, these findings reveal a lipid-mediated regulatory mechanism for intra-Golgi trafficking and establish the critical role of GOLPH3 in vertebrate development.

cell biology↗

bioRxiv2023

A system-wide analysis of lipid transfer proteins delineates lipid mobility in human cells

Lipid transfer proteins (LTPs) maintain the specialised lipid compositions of biological membranes, and many are associated with disease. In eukaryotes, they support organellar functions by transporting lipids between compartmentalised metabolic pathways. However, for the majority of the hundreds of human LTPs, the cargoes remain unknown. We combined biochemical, lipidomic and computational methods to characterize LTP-lipid complexes assembled in cellulo and in an in vitro biochemical assay. We identified bound lipids for about half of the LTPs analysed, and confirmed known cargoes, while discovering new ones for most LTP families. The data represents a systematic resource that captures the general principles of non-vesicular lipid transport in humans. The specificity of LTPs for lipids involves not only the recognition of specific head groups, but also of specific acyl chains. This selectivity defines lipid species within a lipid class with different metabolic or functional fates. The generalised ability of LTPs to form complexes with more than one class of lipids delineates new relationships between lipids and regulatory mechanisms that may contribute to the coordination of metabolism between different organelles. This work represents a resource and a framework for further analyses in different cell types, in pathological states or following various cellular perturbations.

biochemistry↗

bioRxiv2023

Membrane specificity of the human cholesterol transfer protein STARD4

STARD4 regulates cholesterol homeostasis by transferring cholesterol between plasma membrane and endoplasmic reticulum. The STARD4 structure features a helix-grip fold surrounding a large hydrophobic cavity holding the sterol. Its access is controlled by a gate formed by two flexible loops - {Omega}1 and {Omega}4- and the C-terminal -helix. Besides this, little is known about the mechanisms by which STARD4 binds to membranes and extract/releases cholesterol. All available structures of STARD4 are without a bound sterol and display the same closed conformation of the gate. The cholesterol transfer activity of the mouse STARD4 is enhanced in the presence of anionic lipids, and in particular of phosphatidylinositol biphosphates (PIP2) for which two binding sites were proposed on the mouse STARD4 surface. Yet only one of these sites is conserved in human STARD4. We here report the results of a liposome microarray-based assay and microseconds-long molecular dynamics simulations of apo-and holo forms of human STARD4 with complex lipid bilayers mimicking the composition of the donor and acceptor membranes. We show that the binding of apo form of human STARD4 is sensitive to the presence of PIP2 through two specific binding sites, one of which was not identified on mouse STARD4. We report two novel conformations of the gate in holo-STARD4: a yet-unobserved close conformation and an open conformation of {Omega}4 shedding light on the opening/closure mechanism needed for cholesterol uptake/release. Overall, the modulation of human STARD4 membrane-binding by lipid composition, and by the presence of the cargo supports the capacity of human STARD4 to achieve directed transfer between specific organelle membranes.

molecular biology↗

bioRxiv2023

Differential Effects of Posttranslational Modifications on the Membrane Interaction of Huntingtin Protein

Huntingtons disease (HD) is a neurodegenerative disorder caused by an expanded polyglutamine stretch near the N-terminus of the huntingtin (HTT) protein, rendering the protein more prone to aggregate. The first 17 residues in HTT (Nt17) interact with lipid membranes and harbor multiple posttranslational modifications (PTMs) that can modulate HTT conformation and aggregation. In this study, we used a combination of biophysical studies and molecular simulations to investigate the impact of PTMs on the helicity of Nt17 in the presence of various lipid membranes. We demonstrate that anionic lipids such as PI4P, PI(4,5)P2, and GM1 significantly enhance the helical structure of unmodified Nt17. This effect is attenuated by single acetylation events at K6, K9, or K15, whereas tri-acetylation at these sites abolishes Nt17 membrane interaction. Similarly, single phosphorylation at S13 and S16 decreased but did not abolish the POPG and PIP2-induced helicity, while dual phosphorylation at these sites markedly diminished Nt17 helicity, regardless of lipid composition. The helicity of Nt17 with phosphorylation at T3 is insensitive to the membrane environment. Oxidation at M8 variably affects membrane-induced helicity, highlighting a lipid-dependent modulation of Nt17 structure. Altogether, our findings reveal differential effects of PTMs and crosstalks between PTMs on membrane interaction and conformation of HTT. Intriguingly, the effects of phosphorylation at T3 or single acetylation at K6, K9, and K15 on Nt17 conformation in the presence of certain membranes do not mirror that observed in the absence of membranes. Our studies provide novel insight into the complex relationship between Nt17 structure, PTMs and membrane binding. TOC figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/569526v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@153610eorg.highwire.dtl.DTLVardef@10908dforg.highwire.dtl.DTLVardef@256c14org.highwire.dtl.DTLVardef@dac763_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Conformational dynamics of lipid transfer domains provide a general framework to decode their functional mechanism.

Lipid Transfer Proteins (LTPs) are key players in cellular homeostasis and regulation, as they coordinate the exchange of lipids between different cellular organelles. Despite their importance, our mechanistic understanding of how LTPs function at the molecular level is still in its infancy, mostly due to the large number of existing LTPs and to the low degree of conservation at the sequence and structural level. In this work, we use molecular simulations to characterize dynamical and mechanistic aspects of a representative dataset of Lipid Transport Domains (LTDs) of 12 LTPs that belong to 8 distinct families. We find that LTDs display common dynamical, rather than structural, features despite no sequence homology nor structural conservation. These dynamical features correlate with their mechanistic mode of action, allowing to interpret and design experimental strategies to further dissect their mechanism. Our findings indicate the existence of a conserved, fold-independent mechanism of lipid transfer across LTPs of various families and offer a general framework for understanding their functional mechanism.

biophysics↗

bioRxiv2022

Structure and regulation of the Myotonic dystrophy kinase-Related Cdc42-binding Kinase

Remodeling of the cytoskeleton underlies myriad processes essential for life. Protein kinases of the DMPK family are critical regulators of actomyosin contractility in cells. In the nematode worm, Caenorhabditis elegans, MRCK1 is required for the activation of myosin, leading to the development of cortical tension, apical constriction and early gastrulation. Here, we present the structure, conformation, and membrane-binding properties of C. elegans MRCK1. MRCK1 forms an obligate homodimer with N-terminal kinase domains, a parallel coiled-coil of 55 nm, and a C-terminal tripartite module of C1, PH and CNH domains. High-throughput liposome binding assays indicate binding to specific phosphoinositides, while the C-terminal Cdc42-binding (CRIB) motif binds specifically to activated Cdc42. The length of the coiled-coil domain of MRCK, as well as those of the related DMPK kinases ROCK, CRIK and DMPK, is remarkably conserved over millions of years of evolution, suggesting that they may function as molecular rulers to precisely position kinase activity at a fixed distance from the membrane.

biochemistry↗

bioRxiv2019

Structures of three MORN repeat proteins and a re-evaluation of the proposed lipid-binding properties of MORN repeats

MORN (Membrane Occupation and Recognition Nexus) repeat proteins have a wide taxonomic distribution, being found in both prokaryotes and eukaryotes. Despite this ubiquity, they remain poorly characterised at both a structural and a functional level compared to other common repeats. In functional terms, they are often assumed to be lipid-binding modules that mediate membrane targeting. We addressed this putative activity by focusing on a protein composed solely of MORN repeats - Trypanosoma brucei MORN1. Surprisingly, no evidence for binding to membranes or lipid vesicles by TbMORN1 could be obtained either in vivo or in vitro. Conversely, TbMORN1 did interact with individual phospholipids. High- and low-resolution structures of the MORN1 protein from Trypanosoma brucei and homologous proteins from the parasites Toxoplasma gondii and Plasmodium falciparum were obtained using a combination of macromolecular crystallography, small-angle X-ray scattering, and electron microscopy. This enabled a first structure-based definition of the MORN repeat itself. Furthermore, all three structures dimerised via their C-termini in an antiparallel configuration. The dimers could form extended or V-shaped quaternary structures depending on the presence of specific interface residues. This work provides a new perspective on MORN repeats, showing that they are protein-protein interaction modules capable of mediating both dimerisation and oligomerisation.

biochemistry↗