Biology subjects

Gros, P.

Publications and source records attributed to Gros, P..

10 recordsLinked to original sources

bioRxiv2026

USP15 REGULATES NEUROINFLAMMATION AND DRIVES PATHOGENESIS IN SYNUCLEINOPATHIES

Neuroinflammation strongly contributes to the pathogenesis of neurological and neurodegenerative diseases, including Parkinson's disease. We show that ablation of Usp15 in astrocytes and in microglia protects against lethal neuroinflammation in vivo. In a mouse model of synucleinopathy, Usp15 deletion diminishes alpha-syn deposits in the brain, slows disease progression, and increases survival time. The neuroprotective effect of Usp15 is associated with differential expression of inflammatory pathways in situ including interferon stimulated genes. These USP15-dependent effects in vivo are recapitulated in vitro in primary human microglia and astrocytes. We detect high USP15 expression in microglia from Parkinson's patients with strong co-expression with LRRK2 and SNCA. In humans, we detect a strong cis-acting eQTL directing high USP15 expression in CD14+ myeloid cells. The allele driving this eQTL is itself associated with increased disease risk, linking myeloid USP15 expression, elevated USP15 plasma levels in Parkinson's patients, to genetic susceptibility.

neuroscience↗

bioRxiv2026

Identification of a C3b-specific nanobody that does not bind C3 and blocks alternative pathway convertases

The human complement system is a protein network in blood and other body fluids that fights invading pathogens and is involved in maintaining homeostasis. A central step in the complement cascade is the conversion of complement protein C3 to C3b by convertase enzymes. Upon cleavage of C3, the nascent C3b molecule undergoes a large conformational change which exposes new epitopes. Molecules that discriminate between C3b and C3 could provide a powerful tool to selectively bind surface-bound complement activation products while leaving the circulating precursors untouched. In this study, we developed C3b-specific nanobodies by generating phage libraries from llamas immunized with purified and surface-bound C3b. We describe UNbC3b-1 as a high-affinity binder that recognizes soluble and surface-bound C3b molecules but does not bind to C3 in its native state. A 4.2 [A] structure of the UNbC3b-1:C3b complex generated by cryogenic electron microscopy shows that UNbC3b-1 binds C3b at the interface of MG3, MG4, and MG6, overlapping with the complement receptor immunoglobulin (CRIg) binding site. Functionally, we show that UNbC3b-1 inhibits complement activity in the alternative pathway (AP), likely by preventing association between substrate C3 and the AP C3 convertase (C3bBb). Altogether, nanobody UNbC3b-1 is a valuable tool to specifically bind C3b molecules and to selectively inhibit the AP convertases of the complement system.

immunology↗

bioRxiv2025

Tetraspanin CD82 reduces the formation of CADM1 oligomers

CD82 is a member of the tetraspanin protein superfamily and known as a metastasis suppressor. We identified cell adhesion molecule 1 (CADM1) as an interaction partner of CD82. CADM1 mediates cell adhesion by forming cis and trans oligomers that connect membranes. We show that CD82 reduces the formation of CADM1 oligomers when solubilized by detergent, on liposomes and in cellulo using Jurkat T-cells. Our data is consistent with a 1:1 complex of CD82 and CADM1 in cis that leaves the CADM1 trans-interaction site accessible. Cryo-electron microscopy of the CD82:CADM1 heterodimer suggests an interaction site between the large-extracellular loop of CD82 and an Ig-like domain of CADM1. Consistently, liposomes coupled with CADM1 ectodomain show reduced clustering when reconstituted with CD82. We hypothesize that CD82 may affect spacing of the transmembrane helices of CADM1, possibly by interacting with the extracellular Ig-like domains and hence disrupting CADM1 oligomerization between membranes.

biochemistry↗

bioRxiv2025

Structural insights into C3 convertase activity of the classical pathway of complement

Immune protection by the complement system depends on C3 cleavage by C3 convertases that is critical to all three activation pathways. Structural data on convertase formation in the classical pathway and on C3-substrate binding to convertases is lacking. Here, we present the cryo-EM structures of the proconvertase (C4b2), convertase (C4b2b), and convertase-substrate complex (C4b2b-C3) of the classical pathway. The data show that C2 and C4b form proconvertases and convertases like factor B and C3b of the alternative pathway. The substrate C3 binds C4b of the convertase through two interfaces: one also found in the SCIN-inhibited C3bBb dimer, and another facilitated by conformational changes in C3. Bending of C3 and swinging of the C2 protease bring the C3-scissile loop into the active site. The second, charged, C4b-interaction site favors C3- substrate binding, but upon cleavage repels product C3b. Thus, a charge switch-over mechanism effects the catalytic turnover of the convertases producing opsonin C3b.

biochemistry↗

bioRxiv2023

Inhibition of cleavage of human complement component C5 and the R885H C5 variant by two distinct high affinity anti-C5 nanobodies

The human complement system plays a crucial role in immune defense. However, its erroneous activation contributes to many serious inflammatory diseases. Since most unwanted complement effector functions result from C5 cleavage, development of C5 inhibitors, such as clinically approved monoclonal antibody Eculizumab, are of great interest. In this study, we developed and characterized two anti-C5 nanobodies, UNbC5-1 and UNbC5-2. Using surface plasmon resonance (SPR), we determined a binding affinity of 120 pM for UNbC5-1 and 8 pM for UNbC5-2. Competition experiments determined that the two nanobodies recognize distinct epitopes on C5. Both nanobodies efficiently interfered with C5 cleavage in a human serum environment, as they prevented red blood cell lysis via membrane attack complexes (C5b-9) and the formation of chemoattractant C5a. The cryo-EM structure of UNbC5-1 and UNbC5-2 in complex with C5 revealed that the binding interfaces of UNbC5-1 and UNbC5-2 overlap with known complement inhibitors Eculizumab and RaCI3, respectively. UNbC5-1 binds to the MG7 domain of C5, facilitated by a hydrophobic core and polar interactions, and UNbC5-2 interacts with the C5d domain mostly by salt bridges and hydrogen bonds. Interestingly, UNbC5-1 potently binds and inhibits C5 R885H, a genetic variant of C5, that is not recognized by Eculizumab. Altogether, we identified and characterized two different, high affinity nanobodies against human C5. Both nanobodies could serve as diagnostic and/or research tools to detect C5 or inhibit C5 cleavage. Furthermore, the residues targeted by UNbC5-1 hold important information for therapeutic inhibition of different polymorphic variants of C5.

immunology↗

bioRxiv2022

In vivo CRISPR screens reveal SCAF1 and USP15 as novel drivers of pancreatic cancer

Functionally characterizing the genetic alterations that drive pancreatic cancer progression is a prerequisite for Precision Medicine. Here, we developed a somatic CRISPR/Cas9 mutagenesis screen to assess the transforming potential of 125 recurrently mutated long-tail pancreatic cancer genes, which revealed USP15 and SCAF1 as novel and potent Pancreatic ductal adenocarcinoma PDAC tumor suppressors, with USP15 functioning in a haplo-insufficient manner. Mechanistically, we found that loss of USP15 leads to reduced inflammatory responses associated with TNF, TGF-{beta} and IL6 signaling and sensitizes pancreatic cancer cells to PARP inhibition and gemcitabine. Similarly, genetic ablation of SCAF1 reduced inflammatory responses linked to TNF, TGF-{beta} and mTOR signaling and increased sensitivity to PARP inhibition. Furthermore, we identified that loss of SCAF1 resulted in the formation of a truncated inactive USP15 isoform at the expense of full length USP15, functionally coupling SACF1 and USP15. Notably, USP15 and SCAF1 mutations or copy number losses are observed in 31% of PDAC patients. Together, our results demonstrate the utility of in vivo CRISPR to integrate human cancer genomics with mouse modeling to delineate novel cancer driver genes USP15 and SCAF1 such as with potential prognostic and therapeutic implications.

cancer biology↗

bioRxiv2021

A method for intuitively extracting macromolecular dynamics from structural disorder

Macromolecular dynamics manifest as disorder in structure determination, which is subsequently accounted for by displacement parameters (also called temperature factors, or B-factors) or alternate conformations. Though B-factors contain detailed information about structural dynamics, they are the total of multiple sources of disorder, making them difficult to interpret and thus little-used in structural analysis. We report here an approach for decomposing molecular disorder into a parsimonious hierarchical series of contributions, providing an intuitive basis for quantitative structural-dynamics analysis. We demonstrate the decomposition of disorder on example SARS-CoV-2 and STEAP4 structures, from both crystallographic and cryo-electron microscopy data, and reveal how understanding of the macromolecular disorder leads to deeper understanding of molecular motions and flexibility, and suggests hypotheses for molecular mechanisms.

biochemistry↗

bioRxiv2021

C1q binding to surface-bound IgG is stabilized by C1r2s2 proteases

Complement is an important effector mechanism for antibody-mediated clearance of infections and tumor cells. Upon binding to target cells, the antibodys constant (Fc) domain recruits complement component C1 to initiate a proteolytic cascade that generates lytic pores and stimulates phagocytosis. The C1 complex (C1qr2s2) consists of the large recognition protein C1q and a heterotetramer of proteases C1r and C1s (C1r2s2). While interactions between C1 and IgG-Fcs are believed to be mediated by the globular heads of C1q, we here find that C1r2s2 proteases affect the capacity of C1q to form an avid complex with surface-bound IgG molecules (on various DNP-coated surfaces and pathogenic Staphylococcus aureus). The extent to which C1r2s2 contribute to C1q-IgG stability strongly differs between human IgG subclasses. Using antibody engineering of monoclonal IgG we reveal that hexamer-enhancing mutations improve C1q-IgG stability, both in absence and presence of C1r2s2. In addition, hexamer-enhanced IgGs targeting S. aureus mediate improved complement-dependent phagocytosis by human neutrophils. Altogether, these molecular insights into complement binding to surface-bound IgGs could be important for optimal design of antibody therapies.

immunology↗

bioRxiv2020

Implications for tetraspanin-enriched microdomain assembly based on structures of CD9 with EWI-F

Tetraspanins are ubiquitous eukaryotic membrane proteins that contribute to a variety of signaling processes by spatially organizing partner-receptor molecules in the plasma membrane. How tetraspanins bind and cluster partner receptors into so-called tetraspanin-enriched microdomains is unknown. Here we present crystal structures of the large extracellular loop of CD9 in complex with nanobodies 4C8 and 4E8; and, the cryo-EM structure of 4C8-bound CD9 in complex with its prototypical partner EWI-F. The CD9 - EWI-F complex displays a tetrameric arrangement with two centrally positioned EWI-F molecules, dimerized through their ectodomains, and two CD9 molecules, one bound to each EWI-F single-pass transmembrane helix through CD9-helices h3 and h4. In the crystal structures, nanobodies 4C8 and 4E8 bind CD9 at the C and D loop, in agreement with 4C8 binding at the ends of the CD9 - EWI-F cryo-EM complex. Overall, the 4C8 - CD9 - EWI-F - EWI-F - CD9 - 4C8 complexes varied from nearly two-fold symmetric (i.e. with the two CD9 - 4C8 copies in nearly anti-parallel orientation) to ca. 50{degrees} bent arrangements. Since membrane helices h1 and h2 and the EC2 D-loop have been previously identified as sites for tetraspanin homo-dimerization, the observed linear but flexible arrangement of CD9 - EWI-F with potential CD9 - CD9 homo-dimerization at either end provides a new concatenation model for forming short linear or circular assemblies, which may explain the occurrence of tetraspanin-enriched microdomains.

biophysics↗

bioRxiv2020

Structural basis for targeting human cancer antigen STEAP1 with antibodies

Six-transmembrane epithelial antigen of the prostate (STEAP1) is an integral membrane protein that is highly upregulated on the cell surface of several human cancers, making it a promising therapeutic target. It shares sequence homology with three enzymes (STEAP2-4) that catalyze the NADPH-dependent reduction of iron(III). However, STEAP1 lacks an intracellular NADPH-binding domain and does not exhibit cellular ferric-reductase activity. Thus, both the molecular function of STEAP1 and its role in cancer progression remain elusive. Here, we present a ~3.0 [A] cryo-electron microscopy structure of trimeric human STEAP1 bound to three Fab-fragments of the clinically employed antibody mAb120.545. STEAP1 adopts a reductase-like conformation and interacts with the Fabs through its extracellular helices. Enzymatic assays in human cells reveal that STEAP1 promotes iron(III) reduction when fused to the intracellular NADPH-binding domain of its family member STEAP4, implicating STEAP1 as a functional ferric reductase in STEAP hetero-trimers. Our work provides a foundation for deciphering the molecular mechanisms of STEAP1 and will be instrumental in the design of new therapeutic strategies to target STEAP1 in cancer.

biophysics↗