Biology subjects

Fribourg, S.

Publications and source records attributed to Fribourg, S..

4 recordsLinked to original sources

bioRxiv2025

Structural basis for NONO specific modification by the α-chloroacetamide compound (R)-SKBG-1

Among the many proteins involved in cancer progression an increasing number of RNA Binding Proteins (RBPs) are central to the function of a cell and tightly associated to genetic diseases as well as cancer appearance and progression. In a recent study, small molecule inhibitors have been identified as targeting NONO, a RBP known to be involved in mRNA splicing, DNA repair and membraneless organelles stability. Here we report the molecular basis of NONO-targeting by the -chloroacetamide (R)-SKBG-1. We explore the specific binding and enantiomer specificity of NONO towards (R)-SKBG-1 using mass spectrometry and structure determination. We have determined the crystal structure of (R)-SKBG-1-bound to NONO homodimer. This study sheds light on the conformational plasticity of (R)-SKBG-1 when covalently bound to NONO. Altogether these results give an experimental rationale for ligand modification and optimization in a future use as a drug against cancer. SIGNIFICANCEDBHS proteins form a family of three proteins encoded by three different and essential genes. They form obligate homodimers and heterodimers to fulfil their function. In the cell, they are involved in mRNA splicing, DNA repair and membraneless organelles formation. Recently, NONO has been identified as a target of small-molecule inhibitors in prostate cancer cells. Treatment with -chloroacetamide modifies a specific cysteine residue only found in NONO and not in its paralogue proteins SFPQ and PSPC1. Here we provide the molecular basis of -chloroacetamide covalent binding to NONO and we explore the enantiomer specificity of binding. We also demonstrate that -chloroacetamide can target NONO in homodimers and heterodimers and that both binding sites are equivalently modified. Finally, we provide show that -chloroacetamide binding to NONO is driven by the combination of covalent binding and conformational flexibility of the ligand. Altogether, we believe that this study provides useful information for ligand improvement aiming at targeting NONO in cancer cells. OUTLINEO_LINONO residue C145 is targeted by (R)-SKBG-1 C_LIO_LIThe two binding sites are equally modified in NONO homodimers C_LIO_LINONO is specifically targeted and not SFPQ and PSPC1 C_LIO_LI(R)-SKBG-1 adopts multiple conformations in the absence of RNA C_LI

biochemistry↗

bioRxiv2023

Molecular details of the CPSF73-CPSF100 C-terminal heterodimer and interaction with Symplekin

Eukaryotic pre-mRNA is processed by a large multiprotein complex to accurately cleave the 3 end, and to catalyze the addition of the poly(A) tail. Within this cleavage and polyadenylation specificity factor (CPSF) machinery, the CPSF73 endonuclease subunit directly contacts both CPSF100 and the scaffold protein Symplekin to form a subcomplex known as the core cleavage complex (CCC) or mammalian cleavage factor (mCF). Here we have taken advantage of a stable CPSF73-CPSF100 minimal heterodimer from E. cuniculi to determine the solution structure formed by the first and second C-terminal domain (CTD1 and CTD2) of both proteins. We find a large number of contacts between both proteins in the complex, and notably in the region between CTD1 and CTD2. A similarity is also observed between CTD2 and the TATA-box binding protein (TBP) domains. Separately, we have determined the structure of the terminal CTD3 domain of CPSF73, which also belongs to the TBP domain family and is connected by a flexible linker to the rest of CPSF73. Biochemical assays demonstrate a key role for the CTD3 of CPSF73 in binding Symplekin, and structural models of the trimeric complex from other species allow for comparative analysis and support an overall conserved architecture.

biochemistry↗

bioRxiv2019

Dimerization of human Rio2 kinase/ATPase locks its ATP-binding site in an apo state

Rio proteins form a conserved family of atypical protein kinases. Rio2 is a serine/threonine protein kinase/ATPase involved in pre-40S ribosomal maturation. Current crystal structures of archaeal and fungi Rio2 proteins report a monomeric form of the protein. Here, we describe three atomic structures of the human Rio2 kinase showing that it forms a homodimer. Upon self-association, the ATP-binding pocket is hidden from the solvent and the protein is locked in an apo state corresponding to an inactive form of the kinase. The homodimerization is mediated by key residues previously shown to be responsible for ATP binding and catalysis. This unusual protein kinase dimer reveals an intricate mechanism of mutually exclusive substrate binding and oligomeric state formation. We propose that this oligomeric state could serve a dual function in maintaining the protein in an inactive state and being a novel type of nuclear import signal. Significance StatementRio kinases form a family of atypical protein kinases that are believed to be ATPases rather than kinases. The three members of the Rio family are involved in ribosome biogenesis. We show here that contrarily to what was reported so far, Rio2 is able homodimerize in a conformation that locks it in an apo state, preventing its (re)association to pre-mature ribosomes. This unconventional self-association is not seen in any other protein kinase. This mechanism is likely to be transient and could used to efficiently re-import the protein to the nucleus.

biochemistry↗

bioRxiv2018

Varicella-zoster virus CNS vasculitis and RNA polymerase III gene mutation in identical twins

Deficiency in the cytosolic DNA sensor RNA Polymerase III was recently described in children with severe varicella zoster infection in the CNS or lungs. Here we describe a pair of monozygotic female twins, who both experienced severe recurrent CNS vasculitis caused by VZV reactivation. The clinical presentation and findings included recurrent episodes of headache, dizziness, and neurological deficits, cerebrospinal fluid with pleocytosis and intrathecal VZV antibody production, and magnetic resonance scan of the brain showing ischaemic lesions. We performed whole exome sequencing and identified a rare mutation in the Pol III subunit POLR3F. The identified R50W POLR3F mutation is predicted to be damaging by bioinformatics and when tested in functional assays, patient PBMCs exhibited impaired antiviral and inflammatory responses to the PoL III agonist Poly(dA:dT) as well as increased viral replication in patient cells compared to controls. Altogether, these cases add genetic and immunological evidence to the novel association between defects in sensing of AT-rich DNA present in the VZV genome and increased susceptibility to severe manifestations of VZV infection in the CNS in humans.\n\nAbbreviations

immunology↗